Reactive Orange 143

Endpoint summary

Description of key information

The substance was not irritating to skin as well as eyes. The substance was also found not corrosive to skin in an in-vitro skin irrtation test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07February 2012 to 17 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Hygroscopic: Yes, store in well-sealed container
pH: 5.4 at concentration of >80 g/L
Stability in Water: Stability for at least 6 hours at room temperature
Solubility in Water: More than 80 g/L

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: at least 6 weeks old.
- Weight at study initiation: at least 1.0 kg.
- Housing: Animals were housed individually in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy). Hay and wooden sticks were abailable during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70 %, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 07- 17 February 2012

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: The test substance was moistened with 0.5 mL water

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls.

Amount / concentration applied:

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

VEHICLE
The test substance was moistened with 0.5 mL water.

Duration of treatment / exposure:

4 hours

Observation period:

14 days.

Number of animals:

3 males.

Details on 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 one week later, after considering the degree of skin irritation observed in the first animal.

Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm). To facilitate scoring, treated skin areas were re-clipped.
Each animal was treated by dermal application of 0.5 grams of the test substance. The test substance was moistened with 0.5 mL of the vehicle and applied to the skin of one flank, using a metalline patch# of 2x3 cm. The patch was mounted on Micropore tape#, which was wrapped around the abdomen and secured with Coban elastic bandage#.
Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using tap water and watery ethanol (50 % v/v) and/or watery acetone (50 % v/v).

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 404.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Irritation: Four hours exposure to 0.5 g of FAT 40854/A resulted in very slight erythema in one animal on Day 1. In the other animals, erythema could not be scored on Day 1 based on orange/red staining of the treated skin. No oedema was noted in the treated skin areas of the three rabbits. The skin irritation resolved within 24 hours after exposure in all animals.

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

Other effects:

Coloration / Remnants: Orange/red staining of the treated skin by the test substance was observed during the observation period, which did not hamper the scoring of the skin reactions except for two animals on Day 1. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40854/A is not considered as irritant to rabbit's skin.

Executive summary:

In a GLP-compliant study, primary skin irritation/corrosion study with FAT 40854/A in the rabbit (4-hour semi-occlusive application) was carried out according to OECD No.404 and EU method B4. Three rabbits were exposed to 0.5 grams of FAT 40854/A, moistened with water by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure. Exposure to FAT 40854/A resulted in very slight erythema in one animal on Day 1. In the other animals, erythema could not be scored on Day 1 based on orange/red staining of the treated skin. No oedema was noted in the treated skin areas of the three rabbits. The skin irritation resolved within 24 hours after exposure in all animals. There was no evidence of a corrosive effect on the skin. Orange/red staining of the treated skin by the test substance was observed during the observation period, which did not hamper the scoring of the skin reactions except for two animals on Day 1. Based on these results FAT 40854/A is not considered as irritant to rabbit's skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January, 2012 to 20 January, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Control samples:

other: sterile Milli-Q water

Amount/concentration applied:

103.6 mg

Duration of treatment / exposure:

3 minutes and 1 hour
TEST MATERIAL
- Amounts applied: 25 mg solid test substance, 25 μl Milli-Q water to moisten skin.

NEGATIVE CONTOL:
- Amount applied: 50 µl Milli-Q water

POSITIVE CONTROL
- Amount applied: 50 µl
- Concentration: 8N KOH

Duration of treatment / exposure:

3 minutes and 1 hour

Details on study design:

TEST SITE
- EpiDerm Skin Model (EPI-200, Lot no.:16222 kit W). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 3 minutes and 1 hour

POST INCUBATION PERIOD
- Over night at room temperature

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

95

Negative controls validity:

valid

Positive controls validity:

valid

FAT 40854/A 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 FAT 40854/A did not interact with MTT.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40854/A is not corrosive in the in vitro skin corrosion test.

Executive summary:

In a GLP compliant study,ability of FAT 40854/A to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200))an in vitro skin corrosion test with FAT 40854/A according to OECD guideline 431 and EU method B.40. The possible corrosive potential of FAT 40854/A was tested through topical application for 3 minutes and 1 hour. Batch TZ 5719 / BOP 02-11 of FAT 40854/A was a reddish-brown powder. Skin tissue was moistened with 25 μl of Milli-Q water and 25 mg of FAT 40854/A was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 17% after 3 minutes exposure. The absolute mean OD₅₄₀ (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 11 and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 6 %, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with FAT 40854/A compared to the negative control tissues was 86 % and 95 %, respectively. Because the mean relative tissue viability for FAT 40854/A was not below 50% after the 3-minute treatment and not below 15 % after the 1-hour treatment FAT 40854/A is considered to be not corrosive. Based on the test results, FAT 40854/A is not skin corrosive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

13 February 2012 to 19 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Animals used withihn the study were at least 6 weeks old *.
- Weight at study initiation: Body weights were at least 1.0 kg *.
- 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
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 13 February - 19 March 2012

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: average 10.4 mg (range 10.4 – 10.5 mg)

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, 14 and/or 21 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 two weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of, on average, 10.4 mg (range 10.4 – 10.5 mg) 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 (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. Immediately after fluorescein examination on Day 2, the treated eye was rinsed with approximately 50 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove residual test substance. For reference control the other eye was also rinsed. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20 %.

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

OBSERVATIONS
- 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 according to protocol.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and/or 21 days after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Irritation: Instillation of approximately 10 mg of FAT 40854/A (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in all animals. Staining of the sclera was observed and recovered 7 or 14 days after instillation. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2 % fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Corrosion: There was no evidence of ocular corrosion.

Other effects:

Colouration/Remnants: orange/red staining of the eyelids, nictitating membrane and/or sclera was noted immediately after instillation up to and including 7 or 14 days after instillation. Remnants of the test substance were present on the outside of the eyelids on Day 1 up to including 7 or 14 days after instillation. Orange/ red staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40854/A is considered as not irritant to rabbit eye.

Executive summary:

In a GLP-compliant study, an acute eye irritation/corrosion study with FAT 40854/A in the rabbit was carried out based on the OECD guideline 405 and EU method B5. Single samples of approximately 10 mg of FAT 40854/A (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 and 14 and/or 21 days after instillation. Instillation of the test substance resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in all animals. Staining of the sclera was observed and recovered 7 or 14 days after instillation. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2 % fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Orange/red staining of the eyelids, nictitating membrane and/or sclera was noted immediately after instillation up to and including 7 or 14 days after instillation. Remnants of the test substance were present on the outside of the eyelids on Day 1 up to including 7 or 14 days after instillation. Orange/ red staining of the fur on the head and paws, caused by the test substance, was noted during the observation period. Based on these results, FAT 40854/A is considered as not irritant to rabbit eye and does not have to be classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In-vivo Skin Irritation:

In a GLP-compliant study, primary skin irritation/corrosion study with FAT 40854/A in the rabbit (4-hour semi-occlusive application) was carried out according to OECD No.404 and EU method B4. Three rabbits were exposed to 0.5 grams of FAT 40854/A, moistened with water by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure. Exposure to FAT 40854/A resulted in very slight erythema in one animal on Day 1. In the other animals, erythema could not be scored on Day 1 based on orange/red staining of the treated skin. No oedema was noted in the treated skin areas of the three rabbits. The skin irritation resolved within 24 hours after exposure in all animals. There was no evidence of a corrosive effect on the skin. Orange/red staining of the treated skin by the test substance was observed during the observation period, which did not hamper the scoring of the skin reactions except for two animals on Day 1. Based on these resultsFAT 40854/A is not considered as sensitiser to rabbit's skin.

 

In-vitro Skin Irritation:

In a GLP-compliant study, ability of FAT 40854/A to induce skin corrosion on a human three-dimensional epidermal model (EpiDerm (EPI-200)) an in vitro skin corrosion test according to OECD guideline 431 and EU method B.40 was tested. The positive control had a mean relative tissue viability of 17 % after 3 minutes exposure. The absolute mean OD₅₄₀ (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 11 and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 6 %, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with FAT 40854/A compared to the negative control tissues was 86 % and 95 %, respectively. Because the mean relative tissue viability for FAT 40854/A was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment FAT 40854/A is considered to be not corrosive. Based on the test results, FAT 40854/A is not skin corrosive.

 

In vivo eye irritation:

In a GLP-compliant study, an acute eye irritation/corrosion study with FAT 40854/A in the rabbit was carried out based on the OECD guideline 405 and EU method B5. Single samples of approximately 10 mg of FAT 40854/A TE (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 and 14 and/or 21 days after instillation. Instillation of the test substance resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in all animals. Staining of the sclera was observed and recovered 7 or 14 days after instillation. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2 % fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Orange/red staining of the eyelids, nictitating membrane and/or sclera was noted immediately after instillation up to and including 7 or 14 days after instillation. Remnants of the test substance were present on the outside of the eyelids on Day 1 up to including 7 or 14 days after instillation. Orange/ red staining of the fur on the head and paws, caused by the test substance, was noted during the observation period. Based on these results, FAT 40854/A is considered as not irritant to rabbit eye.

 

In-vitro Eye irritation (BCOP):

In a GLP-compliant study, screening for the eye irritancy potential of FAT 40854/A using the Bovine Corneal Opacity and Permeability test (BCOP test) was carried out according to OECD guideline 437 and EU method B. 47. The possible ocular irritancy of FAT 40854/A was tested through topical application for approximately 240 minutes. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and tested neat on top of the corneas (293 to 317 mg). The mean in vitro irritancy score of the negative control was within the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 82 and was within the acceptability criteria of the assay. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The mean in vitro irritancy score was 19 after 240 minutes of treatment with FAT 40854/A. Since the mean in vitro irritancy score for FAT 40854/A was below 55.1 after 240 minutes treatment FAT 40854/A is considered to be not severe irritant or corrosive.

Finally, it is concluded that this test is valid and that FAT 40854/A is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions.

Justification for classification or non-classification

Based on the findings in the skin and eye irritation study, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 to be irritating to the skin as well as eyes.