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Administrative data

Description of key information

Based on an in vitro skin irritation test, conducted according to OECD 439 test guideline and GLP principles, it was concluded that the substance is not irritating to the skin.

Based on an in vivo eye irritation study, conducted in accordance with OECD 405 test guideline and according to GLP principles, it was concluded that the substance is irritating to the eyes.

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:
04-04-2011 to 11-04-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test), adopted 24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37.1 - 37.4
- Humidity (%): 93-95
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 10.2 to 10.4 mg, 5 μl Milli-Q water to moisten skin.

NEGATIVE CONTOL:
- Amount applied: 25 µl phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% sodium dodecyl sulphate in PBS
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Standard ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 11-EKIN-014).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
% tissue viability
Value:
106
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6%
Other effects / acceptance of results:
Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with FDCA compared to the negative control tissues was 106%.

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Mean tissue viability for the test substance was > 50%, therefore the test substance is considered not to be irritant to the skin.

Interpretation of results:
not irritating
Remarks:
According to Regulation (EC) No 1272/2008
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 test guideline and GLP principles.
It is concluded that this test is valid and that FDCA is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) performed according to OECD 439 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (10.2 to 10.4 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 6% whereas the test substance showed cell viability of 106%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that FDCA is not irritating in the current in vitro skin irritation test.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January, 2013 - 12 February, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(2012)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Animals used withihn the study were at least 6 weeks old (actual 9 weeks old)
- Weight at study initiation: Body weights were at least 1.0 kg (actual 2806 - 2808 g)
- Housing: Individually housed in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: average 51.4 mg (range: 50.8 mg – 51.7 mg, approx. 0.1 mL)
Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and 14 days after instillation of the test substance.
Number of animals or in vitro replicates:
3 males
Details on study design:
STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 15 Days later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Both eyes were topically anaesthetized using a local anaesthetic. Approximately 10 minutes prior to treatment, 1 drop was applied to the eyes. No routine pretreatment with a systemic analgesic was done. The subcutaneous injection was
considered to cause unwanted additional distress, since in the majority of cases instillation of test materials does not cause much distress in the topically anesthetized eye. No post-treatment with systemic analgesics were needed.
Animals were treated by instillation of the test substance (a volume of approximately 0.1 mL), 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. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%.

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

OBSERVATIONS (beschrijf beperkt)
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and 14 days after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405.

Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Slight dulling of the normal luster of the cornea, reversible within 72 hours.
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Slight dulling of the normal luster of the cornea, reversible within 48 hours.
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Slight dulling of the normal luster of the cornea, reversible within 48 hours.
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Instillation of approximately 51 mg into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae.
The corneal injury consisted of opacity (maximum grade 1) and epithelial damage (maximum 25, 10 or 10 % of the corneal area). Slight dulling of the normal luster of the cornea was noted up to 48 hours after exposure in all animals. The corneal injury resolved within 72 hours in all animals. Iridial irritation grade 1 was observed after 24 hours and had resolved therafter. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 14 days in all animals.
There was no evidence of ocular corrosion.
Other effects:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.
Interpretation of results:
irritating
Remarks:
According to Regulation (EC) No 1272/2008
Conclusions:
In an eye irritation study with rabbits, performed according to OECD 405 test guidelines and GLP principles, irritation was observed.
Executive summary:

FDCA was tested in an acute eye irritation/corrosion study in 3 male rabbits, performed according to OECD 405 test guidelines and GLP principles.

Instillation of the test substance resulted in effects on the cornea, iris and conjunctivae.

The corneal injury consisted of opacity (maximum grade 1) and epithelial damage (maximum 25, 10 or 10 % of the corneal area). Slight dulling of the normal luster of the cornea was noted up to 48 hours after exposure in all animals. The corneal injury resolved within 72 hours in all animals. Iridial irritation grade 1 was observed after 24 hours and had resolved thereafter. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 14 days in all animals.

There was no evidence of ocular corrosion. No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality

occurred.

Based on these results, the test substance should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-15 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants” (2009)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants" (2008)
Deviations:
no
Qualifier:
according to
Guideline:
other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 124. Bovine Opacity and Permeability Assay - SOP of Microbiological Associates Ltd., 1999
Qualifier:
according to
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992
GLP compliance:
yes
Species:
other: cow
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount applied (weight with unit): approx. 303 to 309 mg per cornea

NEGATIVE CONTROL
- Amount applied (volume with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount applied (volume with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 cornea's
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation).
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by immediate opacity measurement and permeability evaluation of the cornea.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant
Irritation parameter:
in vitro irritation score
Value:
> 26 - < 38
Remarks on result:
other: Time point: 240 min; Mean score of 3 corneas: 34
Interpretation of results:
not irritating
Remarks:
According to Regulation (EC) No 1272/2008
Conclusions:
FDCA is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test

Executive summary:

A Bovine Corneal Opacity and Permeability test was performed according to OECD guideline 437 and GLP principles. The negative and positive control responses of the opacity and permeability values were within the limits of the laboratory historical range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Approx. 303 to 309 mg FDCA were applied per cornea, in total 3 cornea's were tested with the test substance.

The mean in vitro irritancy score was 34 after 240 minutes of treatment with FDCA. Since the mean in vitro irritancy score for FDCA was below 55.1 after 240 minutes treatment FDCA is considered to be not severely irritant or corrosive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) performed according to OECD 439 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin (10.2 to 10.4 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 6% whereas the test substance showed cell viability of 106%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that FDCA is not irritating in the in vitro skin irritation test.

Eye irritation:

A Bovine Corneal Opacity and Permeability test was performed according to OECD guideline 437 and GLP principles.The negative and positive control responses of the opacity and permeability values were within the limits of the laboratory historical range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Approx. 303 to 309 mg FDCA was applied per cornea, in total 3 cornea's were tested with the test substance.

The mean in vitro irritancy score was 34 after 240 minutes of treatment with FDCA. Since the mean in vitro irritancy score for FDCA was below 55.1 after 240 minutes treatment FDCA is considered to be not severely irritant or corrosive.

FDCA was tested in an acute eye irritation/corrosion study in 3 male rabbits, performed according to OECD 405 test guidelines and GLP principles.

Instillation of the test substance resulted in effects on the cornea, iris and conjunctivae.

The corneal injury consisted of opacity (maximum grade 1) and epithelial damage (maximum 25, 10 or 10 % of the corneal area). Slight dulling of the normal luster of the cornea was noted up to 48 hours after exposure in all animals. The corneal injury resolved within 72 hours in all animals. Iridial irritation grade 1 was observed after 24 hours and had resolved thereafter. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 14 days in all animals.

There was no evidence of ocular corrosion. No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Justification for classification or non-classification

Based on the results, the test substance should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation, according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

The test substance does not have to be classified for skin irritation according to Regulation (EC) No 1272/2008.