Registration Dossier

Administrative data

Description of key information

Eye irritation: The substance was considered to be non-irritating to rabbits' eyes. 

Skin: The test substance was considered to be irritating to skin in an in vitro test according to OECD 439. In addition, the test item was non corrosive to skin in an OECD 431 test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-27 to 2015-11-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Delivery date: 17 November 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
- Incubation time:
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
- Concentration: unchanged

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
The colour of the test item/water mixture was observed during the whole incubation period (60 min). The test item and the positive and negative controls were washed off the skin tissues after treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following 68.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
29.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation

Table 1: Results after treatment with the test item and the controls

Dose Group

Exposure

Interval

Absorbance

570 nm

Tissue 1*

Absorbance

570 nm

Tissue 2*

Absorbance

570 nm

Tissue 3*

Mean

Absorbance

of 3 Tissues

Rel. Absor-bance [%] Tissue 1, 2 + 3**

 

Relative standard deviation

[%]

Rel.

Absorbance

[% of

Negative

Control]**

Negative control

60 min

1.402

1.549

1.502

1.484

94.4

104.4

101.2

5.1

100.0

Positive control

60 min

0.066

0.064

0.067

0.066

4.5

4.3

4.5

2.7

4.4

Test item

60 min

0.444

0.433

0.441

0.439

29.9

29.1

29.7

1.4

29.6

* Mean of three replicate wells after blank correction
** relative absorbance per tissue [rounded values]: (100x(absorbance tissue))/ mean absorbance negative control
*** relative absorbance per treatment group [rounded values]: (100x(mean absorbance test item/positive control))/ mean absorbance negative control

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item was found to be irritating to skin according to UN GHS and EU CLP regulation.
Executive summary:

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDermTM were treated with the test item, the negative or the positive control for 60 minutes. 30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS) or the positive control (5 % SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 29.6 % compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50 %. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance is irritating to skin.  

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS GmbH, In den Leppsteinswiesen 19, 64380 Roßdorf
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Human Skin Model (EpiDerm Reconstructed Human Epidermis)
- Tissue batch number: 23308
- Delivery date: 19 January 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 minutes and 60 minutes exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.673 +/- 0.048
- Barrier function: ET-50 = 8.20 hrs
- Contamination: sterile

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 (3 min and 60 min exposure)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL (79.4 µL/cm2)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 min and 1 hour
Number of replicates:
2 tissues (three wells per tissue)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
#1 (3 minutes exposure)
Value:
82.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Remarks:
after 3 minutes exposure
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
#2 (60 min exposure)
Value:
27.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Remarks:
after 1 hour exposure
Interpretation of results:
GHS criteria not met
Conclusions:
The test item Benzyl formate was non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

An in vitro study was performed to assess the corrosive potential of Benzyl formate by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive (colourless). Consequently, additional tests with freeze-killed or viable tissues were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

50 μL of the test item were dispensed directly onto duplicate EpiDermTM tissue surface and spread to match the surface of the tissue. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV [%] in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value decreased to 82.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 27.5%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item Benzyl formate was non corrosive to skin according to EU CLP and UN GHS.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2000-02-28 to 2000-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
, 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rabbit
Strain:
other: SPF albino rabbit
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.2 - 2.6 kg bw
- Housing: The rabbits were caged individually in PPO cages (floor area: 2576 cm²) with perforated floor
- Diet: A pelleted complete rabbit diet "Altromin 2123" from Altromin, D-32791 Lage, Lippe, was available ad libitum.
- Water: The animals had free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth.
- Acclimation period: At least one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 3 °C
- Humidity: 55 +/- 15 %
- Air changes: 10 times/hour
- Photoperiod: 12 hours light and 12 hours darkness (light was on from 6 a.m. to 6 p.m.)
Vehicle:
unchanged (no vehicle)
Controls:
other: the untreated right eye of the same animal served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL


Duration of treatment / exposure:
Single treatment, eyelids were held together for about 1 second.
Observation period (in vivo):
7 days, assessment of damage/irritation was made 1, 24, 48 and 72 hours following treatment.
Number of animals or in vitro replicates:
4
Details on study design:
0.1 mL of the test article was placed in the left eye of the rabbits by gently pulling the lower lid away from the eyeball to form a cup into which the test article was dropped. The lids were then gently held together for 1 second. The eyes were examined and the grade of ocular reaction was recorded one hour and 24 hours later. After the first 24 hour reading Fluorescein was instilled. After rinsing with 20 mL 0.9 % sodium chloride solution the eye were examined again using UV-light to detect possible corneal damage. The eyes were also examined 48 and 72 hours as well as 7 days after the treatment.
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: mean 24, 48, 72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean 24, 48, 72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: mean 24, 48, 72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 d
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
other: mean 24, 48, 72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 d
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: mean 24, 48, 72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: mean 24, 48, 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
other: mean 24, 48, 72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #4
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: mean 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: mean 24, 48, 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
other: mean 24, 48, 72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: mean 24, 48, 72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 d
Remarks on result:
other: Reading after instillation of Fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: mean 24, 48, 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: Reading after instillation of Fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: mean 24, 48, 72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 d
Remarks on result:
other: Reading after instillation of Fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
other: mean 24, 48, 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: Reading after instillation of Fluorescein
Interpretation of results:
GHS criteria not met
Conclusions:
A test was conducted to assess the eye irritating potential of the test item to rabbits' eyes. Four rabbits were used. As a result, the substance was considered to be non irritating to eyes.
Executive summary:

A test was conducted to assess the eye irritating potential ot the test item to rabbits' eyes. Four SPF albino rabbit were used. The study was performed in accordance to GLP and OECD Guideline 405. After a single application of 0.1 mL of the test item, the animals were observed for a period of 7 days and eye reactions were recorded at 1, 24, 48 and 72 hours. The mean scores for cornea opacity calculated for the time points 24, 48 and 72 hours were 0, 0, 0.7 and 0.3 for animal 1, 2, 3 and 4, respectively. Iris mean scores were 0 for each of the four animals. The conjunctiva scores were calculated to be 0.7, 0.7, 1.7, 1.7 and chemosis scores were 0, 0.3, 0.7, 0.3. All findings were reversible within the observation period of 7 days. On the basis of four results, the substance is considered to be non irritating to the rabbits' eyes.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The skin irritation potential of the test item was determined in two in vitro studies according to OECD 431 and 439 and a literature study with guinuea pigs and in human patch tests (see section 7.10.5). The results of these studies will be summarized in the following section.

Skin irritation:
Key Studies
An in vitro study (OECD 439) was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. Each three tissues of the human skin model EpiDermTM were treated with the test item, the negative or the positive control for 60 minutes. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 29.6% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50 %. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance is irritating to skin.


An in vitro study (OECD 431) was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive (colourless). Consequently, additional tests with freeze-killed or viable tissues were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

50 μL of the test item were dispensed directly onto duplicate EpiDermTM tissue surface and spread to match the surface of the tissue. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV [%] in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value decreased to 82.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 27.5%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item was non corrosive to skin according to EU CLP and UN GHS.



Supporting Studies:
Sharp 1978 (Toxicology, 9: 261-271)
A preliminary irritation test was included in a sensitisation study with Hartley strain albino guinea pigs.The irritation potential of the test substance was evaluated after intradermal injection and the topical application of 0.1 mL on the shaved flanks of four animals of the same sex. The reactions were examined 24 h later. After intradermal injection the concentration giving slight but perceptible irritation with no odema was determined to be 0.25 % and after topical application, the highest concentration which caused no irritation was determined to be 20%.

Human Patch tests
Three human dermal patch tests with the test substance are available. The test substance was non-irritating in all three human patch tests (see IUCLID section 7.10.5).

Eye irritation
Key Study
A test was conducted to assess the eye irritating potential of the test item to rabbits' eyes. Four SPF albino rabbit were used. The study was performed in accordance to GLP and OECD Guideline 405. After a single application of 0.1 mL of the test item, the animals were observed for a period of 7 days and eye reactions were recorded at 1, 24, 48 and 72 hours. The mean scores for cornea opacity calculated for the time points 24, 48 and 72 hours were 0, 0, 0.7 and 0.3 for animal 1, 2, 3 and 4, respectively. Iris mean scores were 0 for each of the four animals. The conjunctiva scores were calculated to be 0.7, 0.7, 1.7, 1.7 and chemosis scores were 0, 0.3, 0.7, 0.3. All findings were reversible within the observation period of 7 days. On the basis of four results, the substance is considered to be non irritating to the rabbits' eyes.

Justification for classification or non-classification

The available data gave no indications for an eye-irritating property of the test substance. On the basis of these data the substance is not considered to be classified for eye irritation under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221. The substance is classified as irritating to skin into Category 2 (H315: Causes skin irritation), under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EC) No 2016/1179.