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Description of key information

Skin irritation:
The skin Irritation Potential of Acetamid was determined in the Human Skin Model Test following EU-Method B.46 resp. OECD 439.
Three tissues of the human skin model EpiDermTM were treated with Acetamid for 60 minutes.After the treatment with the test item, the relative absorbance values increased compared to the negative control to 104.9 %. This value is well above the threshold for irritation po-tential (50 %).
Therefore, Acetamid is considered as not skin irritant in the Human Skin Model Test.
Eye irritation:
EPIOCULAR-Test
The Eye Irritation Potential of Acetamid was determined using a Human Cornea Model following Protocol MTT ET-50.
After 3 minutes treatment with the test item, the relative absorbance values were reduced to 76.1 %. After 30 minutes treatment, relative absorbance values were reduced to 71.8 %. And after 60 minutes treatment, the relative absorbance values were reduced to 31.9 %. According to the respective Draft OECD Guideline the test item is identified as potentially requiring classification and labelling according to 13 UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%. Therefore further testing was performed.
BCOP-Test
The test item Acetamid showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0.951. According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

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:
2014-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Data obtained from a guideline study according to OECD Guideline 439 and therefore considered reliable without restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: in vitro
Strain:
other: in vitro
Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
Tissue 1: 26.5 mg
Tissue 2: 27.3 mg
Tissue 3: 26.1 mg
Duration of treatment / exposure:
60 minutes
Number of animals:
3 tissues
Details on study design:
Pre-Tests
First, it was tested whether the test item develops a colour without MTT addition. 24.7 mg were given in a test tube with 0.3 mL H2O demin. and incubated at 37 °C and 5% CO2 for 60 minutes. The resulting solution was colourless, therefore no binding capacity had to be tested.
Then, the test item Acetamid was tested for the ability of direct formazan reduction. To test for this ability, 24.3 mg were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 °C and 5 % CO2 for 60 minutes. Untreated MTT solution was used as control. The MTT solution didn’t change its colour within one hour, therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Pre-Incubation of Tissues
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (up-per row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37 °C and 5 % CO2 for one hour.
After the pre-incubation (one hour), the other three 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 °C and 5 % CO2 for 18 hours.

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 (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer. One plate was used as positive control; each tissue was treated with 30 µL SDS-solution. One plate was used for treatment with the test item. The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.

Tissues were dosed in one minute intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min.. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.
Dosing of first tissue: 11:00 h
Dosing of last tissue: 11:23 h
Start of incubation at 37 °C: 11:25 h
End of incubation at 37 °C: 12:00 h
Rinsing of first tissue: 12:00 h
Rinsing of last tissue: 12:23 h
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). The tissue surfaces were evaluated visually under the stereo microscope, excess test item was removed, where necessary.
Then the tissues were set in the incubator for 24 hours.

Medium Renewal
For three 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 ten min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the in-cubator for 18 ± 2 hours for post-incubation.
The medium from the “old” 6-well-plates was collected in the labelled 24-well-plate. It can be stored for 12 months at – 20 °C for possible interleukin analysis. As the result of the test was unambiguous, the samples will be destroyed after finalisation of the final report.

MTT Assay
After a total incubation time of 42 ± 2 hours, 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 for 3 hours ± 5 min.
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 two hours at room temperature.
After two hours, 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-well-plate which was read in a plate spectral photometer at 570 nm.
Irritation / corrosion parameter:
other: other: % Formazan production
Value:
104.9
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 min. Max. score: 50.0. Reversibility: other: not applicable. (migrated information)

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

Mean Absorption Values

Designation

Negative Control

Acetamid

Positive Control

Mean – blank (Tissue 1)

2.039

2.251

0.045

Mean – blank (Tissue 2)

2.167

2.095

0.047

Mean – blank (Tissue 3) 

1.882

2.038

0.043

Mean of the three Tissues

2.029

2.128

0.045

Relative Standard Deviation
of the three tissues

7.0%

5.2%

4.4%

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

% Formazan Production

Designation

Acetamid

Positive Control

% Formazan production (Tissue 1)

110.9%

2.2%

% Formazan production (Tissue 2)

103.3%

2.3%

% Formazan production (Tissue 3)

100.4%

2.1%

% Formazan production Mean

104.9%

2.2%

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin Irritation Potential of Acetamid was determined in the Human Skin Model Test following EU-Method B.46 resp. OECD 439.
Three tissues of the human skin model EpiDermTM were treated with Acetamid for 60 minutes.After the treatment with the test item, the relative absorbance values increased compared to the negative control to 104.9 %. This value is well above the threshold for irritation po-tential (50 %).

Therefore, Acetamid is considered as not skin irritant in the Human Skin Model Test.
Executive summary:

The skin Irritation Potential of Acetamid was determined in the Human Skin Model Test following EU-Method B.46 resp. OECD 439.

Three tissues of the human skin model EpiDermTMwere treated withAcetamidfor 60 minutes.

In average, 26.6 mg of the solid test item (wetted with 25 µL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by 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. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%).

After the treatment with the test item, the relative absorbance values increased compared to the negative control to 104.9 %. This value is well above the threshold for irritation potential (50 %).

 

Therefore, Acetamid is considered as not skin irritant in the Human Skin Model Test.

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:
2014-12-10 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Data obtained from a guideline study according to the OECD Guideline 437 and therefore considered reliable without restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) in-stead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorption
GLP compliance:
yes (incl. certificate)
Species:
other: not applicable, in vitro
Strain:
other: not applicable, in vitro
Details on test animals or tissues and environmental conditions:
not applicable, in vitro
Vehicle:
other: suspension with a concentration of 20 % in 0.9 % sodium chloride solution
Controls:
other: not applicable, in vitro
Amount / concentration applied:
750 µL of the test item were tested as suspension at 20% concentration in 0.9% sodium chloride solution.
Duration of treatment / exposure:
Exposition time on the corneas was 4 h ± 5 min.
Observation period (in vivo):
330 min
Number of animals or in vitro replicates:
not applicable, in vitro
Irritation parameter:
other: In Vitro Irritancy Score
Basis:
mean
Time point:
other: 4 hours
Score:
> 55
Max. score:
0.951
Reversibility:
not specified

Absorbance and Opacity Values Negative Control

Parameter

Negative Control

Absorbance before exposition

0.1690

0.1633

0.1622

Absorbance after exposition

0.2277

0.2201

0.2861

Opacity before exposition

1.4757

1.4565

1.4528

Opacity after exposition

1.6893

1.6600

1.9324

Opacity Difference

0.2136

0.2035

0.4796

Absorbance and Opacity Values Test Item and Positive Control

Parameter

Test ItemAcetamid 

Positive Control

Absorbance before exposition

0.2165

0.1200

0.1957

0.1634

0.2081

0.1341

Absorbance after exposition

0.2603

0.4343

0.4815

1.8171

1.8312

1.7969

Opacity before exposition

1.6463

1.3183

1.5693

1.4568

1.6147

1.3618

Opacity
after exposition

1.8210

2.7183

3.0304

65.6296

67.7954

62.6470

Opacity
Difference

0.1747

1.4001

1.4611

64.1728

66.1806

61.2852

IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
0.9% NaCl

0.791

0.679

27.2 %

0.466

0.780

Test Item
Acetamid

- 0.129

0.951

103.6 %

1.802

1.180

Positive Control
Imidazole 20%

74.8

77.4

2.9 %

78.3

79.0

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Acetamid showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0.951. According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
Executive summary:

The eye irritative properties of Acetamide were evaluated in the BCOP Test following OECD Guideline 437 resp. EU Method B.47. Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test itemAcetamidwas brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been measured. The test item was incubated on the cornea for4 hours at32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured.

Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea.

20 % imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea.

The test itemAcetamidshowed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0.951.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

No data gaps were identified. The available data are adequate for risk assessment and classification and labelling purposes.


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

Justification for selection of eye irritation endpoint:
The key study is GLP-compliant and of high quality (Klimisch 1).

Justification for classification or non-classification

Skin irritation:

According to CLP; EU GHS (Regulation (EC) No 1272/2008) no classification is required for skin irritation.

Eye irritation:

According to CLP; EU GHS (Regulation (EC) No 1272/2008) no classification is required for eye irritation.