Registration Dossier

Administrative data

Description of key information

Skin:

In the in vitro skin irritation test using the EpiDerm human epidermis model 25 mg test item + 25 µL DPBS were applied topically for 60 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 50% (137%) after 60 min treatment and 42 h post incubation.

Eye:

The tes item did not show corrosive effects in an in vitro ocular model (BCOP).

In an in vivo study in rabbits only temporary slight irritation was found in the conjunctivae. only 1 of 3 animals showed finings of the cornea and iris. All animals recovered completely within 4 to 9 days.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro (Epiderm)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: EU Method b.46 (Skin Irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Species:
other: EpiDerm reconstructed human epidermis model (MatTek Corp.)
Details on test animals and environmental conditions:
organotypic reconstructed three-dimensional model of the human epidermis
Type of coverage:
other: direct application
Controls:
other: three tissues for negative control (30µL DPBS) and positive control (30µL 5% SDS)
Amount / concentration applied:
solids: 25 µL DPBS + 25 mg
Duration of treatment / exposure:
60 +/- 1 minutes
Observation period:
42 +/- 4 h
Number of animals:
three replicate tissues per dose group
Details on study design:
3 replicate tissues are dosed with the test item, the negative control (DPBS) and the positive control (5% SDS), respectively. The tissues are incubated for 35 +/-1 minutes in a humidified incubator (37+/- 1 °C, 5 +/- 1% CO2, 95% RH) and further 25 minutes at room temperature. Then the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 24 +/- 2 h and, after medium renewal, for further 18 +/- 2 h. Then the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 550 nm.
Irritation parameter:
other: mean relative tissue viability
Basis:
other: mean tissue viability of the negative control tissues
Time point:
other: 60 min treatment 42 h post-incubation
Score:
> 50
Remarks on result:
other: non-irritant; EU CLP and UN GHS: No Category
Irritation parameter:
other: mean relative tissue viability
Basis:
other: mean tissue viability of the negative control tissues
Time point:
other: 60 min treatment 42 h post-incubation
Score:
<= 50
Remarks on result:
other: irritant; EU CLP and UN GHS Category 2
Irritant / corrosive response data:
If mean tissue viability is > 50% relative to the mean negative control, the test item is classified as non-irritant (EU CLP and UN GHS: No Category).
If mean tissue viability is =< 50% relative to the mean negative control, the test item is classified as irritant (EU CLP and UN GHS: Category 2).
Other effects:
The test item showed no direct MTT reducing capability and no colouring potential.

Name

NC

PC

TM

Tissue

1

2

3

1

2

3

1

2

3

OD550(blank-corrected)

1.320

2.141

1.843

0.123

0.117

0.117

1.961

2.564

2.449

1.626

1.912

1.814

0.137

0.089

0.121

2.566

2.601

2.443

mean OD550of the replicates (blank-corrected)

1.473

2.026

1.828

0.130

0.103

0.119

2.263

2.582

2.446

total mean OD550of 3 replicate tissues (blank-corrected)

1.776

0.117

2.430

SD OD550

 

0.279

 

 

0.016

 

 

0.239

 

relative tissue viability [%]

82.9

114.1

103.0

7.3

5.8

6.7

127.5

145.4

137.7

SD tissue viability [%]***

15.8

0.8

9.0

CV [% viability]

 

15.8

 

 

11.5

 

 

6.6

 

mean relative tissue viability [%]

100.0

6.6

136.9

*          Corrected mean OD550of the negative control corresponds to 100% absolute tissue viability.

**      mean relative tissue viability of the three positive control tissues is < 20%

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

Interpretation of results:
not classified
Remarks:
Migrated information non-irritant Criteria used for interpretation of results: EU
Conclusions:
The test item is classified as "non-irritant" (EU CLP and UN GHS: No Category).
Executive summary:

In the in vitro skin irritation test using the EpiDerm human epidermis model 25 mg test item + 25 µL DPBS were applied topically for 60 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 50% (137%) after 60 min treatment and 42 h post incubation.

This study is classified as acceptable:

Mean OD550 of the three negative control tissues is >=1 and <=2.5.

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

Standard deviation (SD) of relative tissue viablity obtained from each three concurrently tested tissues is < 18%.

This study satisfies the requirement for Test Guideline OECD 439 for in vitro skin irritation.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-25 to 2012-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)
Vehicle:
physiological saline
Amount / concentration applied:
The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.
Duration of treatment / exposure:
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.
Details on study design:
Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals (from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)
Irritation parameter:
in vitro irritation score
Value:
ca. 1.39
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The eye irritancy potential of Fatty acids, C16-18-, reaction products with diethanolamine was investigated in the bovine corneal opacity
and permeability assay.
The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

The following mean in vitro irritation score was calculated:
1.39
Therefore the test item was non irritant to eyes.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean
and therefore this assay is considered to be valid.

Table:   Opacity

 

Cornea No.

 

Test Item

 

Initial Opacity

 

Final Opacity

 

Change of Opacity Value

 

Corrected Opacity Value

 

1

Negative control

 

4

3

-1

 

2

4

5

1

 

3

4

5

1

 

MV

4,00

4,33

0,33

 

4

Positive control

5

174

169

168,67

5

5

172

167

166,67

6

5

178

173

172,67

MV

5,00

174,67

169,67

169,33

7

Test item

3

5

2

1,67

8

3

4

1

0,67

9

3

4

1

0,67

MV

3,00

4,33

1,33

1,00

 

 

 Table:   Permeability

 

Cornea No.

 

Test Item

 

OD490

 

Corrected OD490 Value

 

1

Negative control

 

0,031

 

2

0,029

 

3

0,027

 

MV

0,029

 

4

Positive control

2,051

2,022

5

2,098

2,069

6

2,123

2,094

MV

2,091

2,062

7

Test item

0,081

0,052

8

0,053

0,024

9

0,031

0,002

MV

0,055

0,026

 

 

Table:   In Vitro Irritation Score

 

Cornea No.

 

Test Item

 

Corrected Opacity Value

 

Corrected OD490 Value

 

IVIS

 

1

Negative control

 

-1,00

0,031

 

2

1,00

0,029

 

3

1,00

0,027

 

MV

0,33

0,029

0,77

4

Positive control

168,67

2,022

 

5

166,67

2,069

 

6

172,67

2,094

 

MV

169,33

2,062

200,26

7

Test item

1,67

0,052

 

8

0,67

0,024

 

9

0,67

0,002

 

MV

1,00

0,026

1,39

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
According to the evaluation criteria the test item Fatty acids, C16-18-, reaction products with diethanolamine is considered as non irritant to eyes.
Executive summary:

Summary Results

The eye irritancy potential of Fatty acids, C16-18-, reaction products with diethanolamine was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item:                     diluted with physiological saline 0.9% NaCl to gain a 20% concentration

Mean in vitro irritation score:                  1.39

Evaluation: non irritant

 

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

 

Conclusion

According to the evaluation criteria the test item Fatty acids, C16-18-, reaction products with diethanolamine is considered as non irritant to eyes.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-27 to 2013-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Test System

Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: female
Body weight at the
beginning of the study: > 2kg
Age at the
beginning of the study: approximately 18 - 24 weeks old
Number of animals: 3
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions

- Semi barrier in an air-conditioned room
- Temperature: 18 +/- 3 °C (recommendations of TVT , GV-SOLAS )
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits (lot no. 1516), rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Housed in ABS - plastic rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
A dose of 0.1 g of the test item was applied to the test site.
Duration of treatment / exposure:
The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball.
The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as
control.
To remove test substance residues the treated eye was rinsed with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 121678081, expiry date: 03/2015) in animals no. 1 and 3 24 hours after the application. The treated eye of animal no. 2 was not rinsed after the application as it was not considered necessary.
Observation period (in vivo):
The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period was extended up to 9 days after dosing.
Number of animals or in vitro replicates:
The in vivo test was performed initially using one animal.
The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described.
In order to confirm the response, two additional animals were treated in the same manner.
Details on study design:
Preparation of the Animals

Approximately 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was
performed to ensure the good state of health of the animals.
Approximately 24 hours before the application the eyes were also examined with the aid of a fluorescein solution (Fluoreszein SE Thilo,
Alcon Pharma, lot no. H 201, expiry date: 12/2013). The eyes were rinsed with physiological saline 0.9% NaCl (B. Braun Melsungen,
lot no. 121678081, expiry date: 03/2015) after the examination.
None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Application

One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no.: 5316, expiry date: 12/2016) was
administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approximately 5 minutes prior to the application of
the test item, 2-3 drops of an ocular anaesthetic (proparacaine hydrochloride ophtalmic 0.5% solution, Ursapharm, lot no.: 273851,
expiry date: 05/2013) were administered in both the treated and the control eye of each animal.
The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball.
The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as
control.
To remove test substance residues the treated eye was rinsed with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 121678081,
expiry date: 03/2015) in animals no. 1 and 3 24 hours after the application. The treated eye of animal no. 2 was not rinsed after the application as it was not considered necessary.
Animals showing pain or distress after the application of the test item were treated with the following doses of buprenorphine:
Animal No. Dose Administration
1 1 ampulla (0.3 mg) of buprenorphine via 600 mL drinking water
Based on an average daily water intake from 15 to 50 mL/kg body weight, buprenorphine was administered at a dose range from 0.0075 to
0.025 mg/kg body weight, diluted in water.

Clinical Observation

The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according to the
grades in the table below.
For the calculation only the 24, 48 and 72-hour readings were used.
72 hours post-application as well as at the end of the prolonged observation period the treated eyes were examined with the aid of a fluorescein
solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.


Evaluation of Results

Individual reactions of each animal were recorded at each time of observation.
Nature, severity and duration of all lesions observed were described.
For the calculation only the 24, 48 and 72-hour readings were used.
On the basis of the test results, the test substance may be classified in any of the following classes in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC :
R36 Irritating to eyes
- Substances and preparations which, when applied to the eye of the animal, cause significant ocular lesions which occur within 72 hours after exposure and which persists for at least 24 hours.
Ocular lesions are significant if the mean scores of the eye irritation test cited in Annex B.5 to Commission Regulation (EC) No. 440/2008, L 142
have any of the following values:
- corneal opacity ≥ 2 but < 3,
- iris lesion ≥ 1 but ≤ 1.5,
- redness of the conjunctiva ≥ 2.5,
- oedema of the conjunctiva (chemosis) ≥ 2,
or, in case where Annex B.5 test have been completed using three animals if the lesions, on two or more animals, are equivalent to any of the above
values except that for iris lesion the value should be ≥ 1 but < 2 and for redness of the conjunctiva the value should be ≥ 2.5.
In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be used in calculating the respective mean values.
- Substances or preparations which cause significant ocular lesions, based on practical experience in humans.
- Organic peroxides except where evidence to the contrary is available.
R41 Risk of serious damage to eyes
Substances and preparations which, when applied to the eye of the animal cause severe ocular lesions which occur within 72 hours after exposure and which persist for at least 24 hours.
Ocular lesions are severe if the means of the scores of the eye irritation test cited in Annex B.5 to Commission Regulation (EC) No. 440/2008, L 142 have any of the values:
- cornea opacity ≥ 3,
- iris lesion > 1.5.
The same shall be the case where the test has been completed using three animals if these lesions, on two or more animals, have any of the values:
- cornea opacity ≥ 3,
- iris lesion = 2.
In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be used in calculating the respective mean values.
Ocular lesions are also severe when they are still present at the end of the time of observation.
Ocular lesions are also severe if the substance or preparation causes irreversible colouration of the eyes.
- Substances and preparations which cause severe ocular lesions, based on practical experience in humans.
Note:
When a substance or preparation is classified as corrosive and assigned R34 or R35, the risk of severe damage to eyes is considered implicit and R41 is not included in the label.
On the basis of the test results, following risk phrases shall be assigned in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008:
Eye irritant Category 1:
Irreversible effects on the eyes / serious damage to the eyes
An eye irritant category 1 is a test material that produces:
- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or that have not fully reversed within 21 days; and/or
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 3; and/or
(ii) iritis > 1.5
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material.
Eye irritant Category 2:
Reversible effects on the eyes / Irritating to the eyes
An eye irritant category 2 is a test material that produces:
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 1; and/or
(ii) iritis ≥ 1; and/or
(iii) conjunctival redness ≥ 2; and/or
(iv) conjunctival oedema (chemosis) ≥ 2
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material, and which fully reverse within an observation period of 21 days.
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in GHS - Globally Harmonized System of Classification and Labelling of Chemicals, third revised edition, July 2009:
Category 1: An eye irritant Category 1 (irreversible effects on the eye) is a test material that produces:
- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or that have not fully reversed within 21 days; and/or
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 3; and/or
(ii) iritis > 1.5
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material.
Category 2A: An eye irritant Category 2A (irritating to eyes) is a test material that produces:
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 1; and/or
(ii) iritis ≥ 1; and/or
(iii) conjunctival redness ≥ 2; and/or
(iv) conjunctival oedema (chemosis) ≥ 2
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material, and which fully reverse within
21 days.
Category 2B: Within this category an eye irritant is considered mildly irritating to eyes (Category 2B), when the effects listed above are fully reversible within 7 days of observation.
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 8 d
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.33
Max. score:
4
Reversibility:
fully reversible within: 6 d
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 5 d
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 5 d
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.67
Max. score:
3
Reversibility:
fully reversible within: 9 d
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
cornea opacity score
Basis:
animal: #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 4 d
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritant / corrosive response data:
After the application into the eyes of three female NZW rabbits the test item produced irritant but not corrosive effects in all animals.
Neither mortalities nor significant clinical signs of toxicity were observed.
The eyes were rinsed after the application in animals no. 1 and 3 to remove test item residues but were not rinsed in animal no. 2 as it was not
considered necessary.
Upon fluorescein examinations 72 hours after application slight corneal lesions were found in animal no. 1. Upon fluorescein examinations at the
end of the prolonged observation period no corneal lesions were found in any animal.
Conjunctival redness, chemosis and discharge were observed in all animals, corneal effects and iris lesion were additionally observed in animal
no. 1 and are individually described.
Other effects:
Body Weight Development

There were no significant body weight changes during the observation period.

Eye Irritation Scores

Animal No. 1

Single Data

 

Time Post-Application

Average Score
(24, 48 and 72 hours)

1 hour

24 hours

48 hours

72 hours

T/C

T/C

T/C

T/C

Conjunctival Redness

2/0

2/0

2/0

2/0

2.00

Conjunctival Chemosis

2/0

3/0

2/0

2/0

2.33

Iris

0/0

1/0

1/0

1/0

1.00

Cornea

0/0

1/0

1/0

1/0

1.00

Single Data - Prolonged Observation Period

 

Time Post-Application

4 days

5 days

6 days

7 days

8 days

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

2/0

2/0

1/0

1/0

0/0

Conjunctival Chemosis

2/0

1/0

0/0

0/0

0/0

Iris

1/0

0/0

0/0

0/0

0/0

Cornea

1/0

0/0

0/0

0/0

0/0

Animal No. 2

Single Data

 

Time Post-Application

Average Score
(24, 48 and 72 hours)

1 hour

24 hours

48 hours

72 hours

T/C

T/C

T/C

T/C

Conjunctival Redness

2/0

2/0

2/0

1/0

1.67

Conjunctival Chemosis

2/0

2/0

1/0

1/0

1.33

Iris

0/0

0/0

0/0

0/0

0.00

Cornea

0/0

0/0

0/0

0/0

0.00

Single Data - Prolonged Observation Period

 

Time Post-Application

4 days

5 days

6 days

7 days

8 days

9 days

T/C

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

1/0

1/0

1/0

1/0

1/0

0/0

Conjunctival Chemosis

0/0

0/0

0/0

0/0

0/0

0/0

Iris

0/0

0/0

0/0

0/0

0/0

0/0

Cornea

0/0

0/0

0/0

0/0

0/0

0/0

Animal No. 3

Single Data

 

Time Post-Application

Average Score

(24, 48 and 72 hours)

1 hour

24 hours

48 hours

72 hours

T/C

T/C

T/C

T/C

Conjunctival Redness

1/0

1/0

1/0

1/0

1.00

Conjunctival Chemosis

1/0

1/0

0/0

0/0

0.33

Iris

0/0

0/0

0/0

0/0

0.00

Cornea

0/0

0/0

0/0

0/0

0.00

Single Data - Prolonged Observation Period

 

Time Post-Application

4 days

 

 

 

 

T/C

 

 

 

 

Conjunctival Redness

0/0

 

 

 

 

Conjunctival Chemosis

0/0

 

 

 

 

Iris

0/0

 

 

 

 

Cornea

0/0

 

 

 

 

T = test item,      C = control Clinical Signs of Toxicity

Animal No. 1

Time after
Test Item Application

Systemic Findings

Local Findings
(apart from findings in Table 3)

1 hour

nsf

discharge grade 2

24 hours

nsf

discharge grade 3

48 hours

nsf

discharge grade 1

72 hours

nsf

discharge grade 1

4 days

nsf

discharge grade 1

5 days

nsf

discharge grade 1

 6 days

nsf

nsf

7 days

nsf

nsf

8 days

nsf

nsf

Animal No. 2

Time after
Test Item Application

Systemic Findings

Local Findings
(apart from findings in Table 4)

1 hour

nsf

discharge grade 2

24 hours

nsf

discharge grade 2

48 hours

nsf

discharge grade 1

72 hours

nsf

nsf

4 days

nsf

nsf

5 days

nsf

nsf

 6 days

nsf

nsf

7 days

nsf

nsf

8 days

nsf

nsf

9 days

nsf

nsf

Animal No. 3

Time after
Test Item Application

Systemic Findings

Local Findings
(apart from findings in Table 5)

1 hour

nsf

discharge grade 1

24 hours

nsf

nsf

48 hours

nsf

nsf

72 hours

nsf

nsf

4 days

nsf

nsf

nsf: no specific finding Absolute Body Weights in kg

 

Animal No. 1

Animal No. 2

Animal No. 3

Start of Study
(weight in kg)

4.0

5.1

4.9

72 Hours Post-Application
(weight in kg)

4.0

5.2

5.0

End of Observation Period
(weight in kg)

4.0

5.3

5.0

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, a single ocular application of the test item Fatty acids, C16-18-, reaction products with diethanolamine to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within up to 9 days. Neither mortalities nor significant clinical
signs of toxicity were observed.
In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according to Annex VI of Commission Directive 2001/59/EC , the test item Fatty acids, C16-18-, reaction products with diethanolamine has no obligatory labelling
requirement for eye irritation.
According to Annex I of Regulation (EC) 1272/2008, the test item Fatty acids, C16-18-, reaction products with diethanolamine has no obligatory
labelling requirement for eye irritation.
According to GHS (Globally Harmonized Classification System) the test item Fatty acids, C16-18-, reaction products with diethanolamine has no
obligatory labelling requirement for eye irritation.
For details of the classification criteria see Evaluation of Results.
Executive summary:

 Summary Results

Species/strain:                   New Zealand White Rabbits Crl: KBL (NZW)

Number of animals:          3

Amount of substance:      0.1 g per test site (neat)

First time of effects:         animals no. 1 and 2:1 hour post-application redness grade 2 and chemosis grade 2 and discharge grade 2

animal no. 3: 1 hour post-application redness grade 1, chemosis grade 1 and discharge grade 1       

Last time of effects:         animal no. 1: 7 days post-applicationredness grade 1

animal no. 2: 8 days post-applicationredness grade 1

animal no. 3: 72 hours post-applicationredness grade 1

Reversibility of

the observed effects:        animal no. 1: the changes were fully reversible within 8 days post-application

animal no. 2: the changes were fully reversible within 9 days post-application

animal no. 3: the changes were fully reversible within 4 days post-application

Method:                           OECD 405

EC 440/2008, Method B.5
OPPTS 870.2400

Thecalculated mean scores did not exceed the limit values according to Directive 2001/59/EC, to Regulation (EC) 1272/2008 and to GHS (for details of the classification criteria see Evaluation of Results).

Mean Values of Eye Irritation Scores – (24, 48, 72 Hour Reading)

Animal No.

Sex

Conjunctival Redness

Conjunctival Chemosis

Iris

Cornea Opacity

1

female

2.00

2.33

1.00

1.00

2

female

1.67

1.67

0.00

0.00

3

female

1.00

0.33

0.00

0.00

 


 

Conclusion

Under the conditions of the present study, a single ocular application of the test item Fatty acids, C16-18-, reaction products with diethanolamine to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within up to 9 days. Neither mortalities nor significant clinical signs of toxicity were observed.

In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according toAnnex VI of Commission Directive 2001/59/EC, the test item Fatty acids, C16-18-, reaction products with diethanolaminehas no obligatory labelling requirement for eye irritation.

According to Annex I of Regulation (EC) 1272/2008, the test item Fatty acids, C16-18-, reaction products with diethanolamine has no obligatory labelling requirement for eye irritation.

According to GHS (Globally Harmonized Classification System) the test item Fatty acids, C16-18-, reaction products with diethanolamine has no obligatory labelling requirement for eye irritation.

For details of the classification criteria see Evaluation of Results.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

skin:

In the in vitro skin irritation test using the EpiDerm human epidermis model 25 mg test item + 25 µL DPBS were applied topically for 60 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 50% (137%) after 60 min treatment and 42 h post incubation.

This result was considered as indication of no irritant potential of the test item on the skin.

Eye:

The tes item did not show corrosive effects in an in vitro ocular model (BCOP).

In an in vivo study in rabbits only temporary slight irritation was found in the conjunctivae. only 1 of 3 animals showed finings of the cornea and iris. All animals recovered completely within 4 to 9 days.

This result was considered as indication of no significant irritant potential of the test item on eyes and mucous membranes.

Justification for classification or non-classification

Not classified.

Skin: The test item showed no irritant effects in an in vitro human skin model according to OECD guideline 439.

Eye: The test item did not show corrosive effects in an in vitro ocular model (BCOP) and the mild conjunctival irritation in an in vivo eye irritation study in rabbits were not sufficient for classsification.