Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

Endpoint summary

Description of key information

Skin:

In an in vitro study in reconstructed human skin (OECD439), the test substance did not show skin irritating effects.

The mean relative tissue viability for C.I. Leuco Sulphur Brown 96 was 98% after 15 minutes treatment.

 

Eye:

In an in vitro eye irritation study (OECD 437) an In Vitro Irritancy Score (IVIS) of -0.4 was determined after 10 minutes treatment, indicating no irritant effects.

In an eye irritation study with rabbits (OECD 405), instillation of the test substance resulted in irritation of the conjunctivae, consisting of redness, chemosis and discharge, which had completely resolved within 72 hours in all animals. The observed slight irritation, was insufficient for classification.

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:

18-Jun-2012 to 10-Sep-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The skin was moistened with 5 µl Milli-Q water to ensure close contact of the test substance to the tissue and the pasty test substance was added on top of the skin tissues using a cotton swab. Consequently, the exact amount of test substance applied was unknown.

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure:15 minutes
Post incubation period: 42 hours

Details on study design:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

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

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. 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:

other: percentage viability

Remarks:

percentage of control / time point: 15 minutes

Value:

98

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: C.I. Leuco Sulphur Brown 96 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 C.I. Leuco Sulphur Brown 96 did not interact with MTT.

The positive control had a mean cell viability after 15 minutes exposure of 46% and did not meet the acceptability criteria. Approximately 1 hour after the performance of this test another in vitroskin irritation test was performed using the same batch of skin and the same batch of 5% (aq) SDS. The positive control had a mean cell viability of 5% and met the acceptability criteria. The absolute mean OD₅₇₀of the negative control tissues (in both in vitro skin irritation tests) were within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly. It can be concluded that the deviation in the mean cell viability of the positive control in the first in vitro skin irritation test was caused by a technical error.

Interpretation of results:

not irritating

Conclusions:

Based on an in vitro skin irritation test, conducted according to OECD 439 guideline, the test item is considered not to be irritant.

Executive summary:

An in vitro test in reconstructed human skin (OECD 439) was performed to assess the irritation/corrosion potential of the test item. Twenty-five µL of the test substance were applied to the skin for 15 minutes. After test substance removal and a post incubation period of 42 hours the cytotoxicity was determined by means of Formazan reduction to MTT.

The relative mean tissue viability obtained after 15 minutes treatment with C.I. Leuco Sulphur Brown 96 compared to the negative control tissues was 98%. Since the mean relative tissue viability for C.I. Leuco Sulphur Brown 96 was above 50% after 15 minutes treatment C.I. Leuco Sulphur Brown 96 is considered to be non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 - 22 November 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 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

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 (8-11 weeks).
- Weight at study initiation: Body weights were at least 1.0 kg (1966 - 2386 grams). Body weight of one animal was determined two days prior to treatment instead of on the day of treatment.
- 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.

IN-LIFE DATES: From: 15 to 22 November 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 (weight with unit):
average 100 mg (range 99.5 – 100.5 mg) (a volume of approximately 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 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 4 Days later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of, on average, 100 mg (range 99.5 – 100.5 mg) of the test substance as the maximum required amount 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.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: two animals: Day of treatment (prior to instillation) and after the final observation.
one animal: Two days prior to treatment (see Protocol Deviations) 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 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.

Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp. In cases of equivocal results when comparing the treated and untreated eyes, the illustrated guide from the Consumer Product Safety Commission, Washington, D.C. 20207 was used for additional control purposes.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

(redness]

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Instillation of approximately 100 mg of C.I. Leuco Sulphur Brown 96 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 72 hours in all animals.

No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

There was no evidence of ocular corrosion.

Other effects:

Colouration/Remnants: Remnants of the test substance were present in the eye of all animals on Day 1.
Brown/black staining of the fur on the head and paws, caused by the test substance, was noted for all animals during the observation period.


Toxicity/Mortality: 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:

In an eye irritation study with rabbits, performed according to OECD guideline 405, instillation of the test substance resulted in irritation of the conjunctivae, consisting of redness, chemosis and discharge, which had completely resolved within 72 hours in all animals. The observed slight irritation, was insufficient for classification.

Executive summary:

Approximately 100 mg of C.I. Leuco Sulphur Brown 96 were instilled into one eye of each of three male rabbits. The other eye remained untreated and served as a control. The eyes were examined approximately 1, 24, 48 and 72 hours after test substance installation. Irritation of the conjunctivae, which consisted of redness, chemosis and discharge (mean score per animal: 0.7) were observed. The slight irritation had completely resolved within 72 hours in all animals. No iridial irritation or corneal opacity and no corneal epithelial damage was observed. There was no evidence of ocular corrosion.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): C.I. Leuco Sulphur Brown 96 was applied directly on the corneas in such a way that the cornea was completely covered by using a cotton bud

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

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 10% (w/v) Benzalkonium Chloride

Duration of treatment / exposure:

- Exposure: 10 minutes
- Post incubation period: 120 minutes

Details on study design:

TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

- 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

Remarks:

time point: 10 minutes

Value:

-0.4

The negative control responses for opacity and permeability were less than the upper limits of 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 (10% (w/v) Benzalkonium Chloride) was 126 and was within the historical positive control data range.It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro Irritancy Score (IVIS) of the test item was determined to be -0.4 after 10 minutes treatment, indicating no irritant effects.

Executive summary:

Bovine corneae were exposed to the test substance for 10 minutes with subsequent rinsing to remove the substance and further incubation for 2 hours with fresh medium. The opacity was measured with an opacitometer and the permeability of the corneae was determined after incubation with sodium fluorescein (90 minutes) by a microplate reader.

The mean opacity and mean permeability values (OD490) of each treatment group were used for the calculation of the In Vitro Irritancy Score (IVIS) according to the following formula: IVIS = mean opacity value + (15x mean OD490 value).

The negative control responses for opacity and permeability indicated that the negative control did not induce irritancy on the corneas. The IVIS of the positive control showed a clear positive effect providing evidence for a valid test performance.

C.I. Leuco Sulphur Brown 96 did not induce ocular irritation through both endpoints, resulting in a mean IVIS of -0.4 after 10 minutes of treatment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

An in vitro test in reconstructed human skin (OECD 439) was performed to assess the irritation/corrosion potential of the test item. Twenty-five µL of the test substance were applied to the skin for 15 minutes. After test substance removal and a post incubation period of 42 hours the cytotoxicity was determined by means of Formazan reduction to MTT.

The relative mean tissue viability obtained after 15 minutes treatment with C.I. Leuco Sulphur Brown 96 compared to the negative control tissues was 98%. Since the mean relative tissue viability for C.I. Leuco Sulphur Brown 96 was above 50% after 15 minutes treatment C.I. Leuco Sulphur Brown 96 is considered to be non-irritant.

Eye:

The potential of the test substance to induce eye irriation/corrosion was investigated in vitro using a Bovine Corneal Opacity and Permeability Test (BCOP) according to OECD 437 and an in vivo eye irritation test in rabbits (OECD 405).

BCOP (OECD 437)

Bovine corneae were exposed to the test substance for 10 minutes with subsequent rinsing to remove the substance and further incubation for 2 hours with fresh medium. The opacity was measured with an opacitometer and the permeability of the corneae was determined after incubation with sodium fluorescein (90 minutes) by a microplate reader.

The mean opacity and mean permeability values (OD490) of each treatment group were used for the calculation of the In Vitro Irritancy Score (IVIS) according to the following formula: IVIS = mean opacity value + (15x mean OD490 value).

The negative control responses for opacity and permeability indicated that the negative control did not induce irritancy on the corneas. The IVIS of the positive control showed a clear positive effect providing evidence for a valid test performance.

C.I. Leuco Sulphur Brown 96 did notinduce ocular irritation through both endpoints, resulting in a mean IVIS of -0.4 after 10 minutes of treatment.

In vivo (OECD 405)

Approximately 100 mg of C.I. Leuco Sulphur Brown 96 were instilled into one eye of each of three male rabbits. The other eye remained untreated and served as a control. The eyes were examined approximately 1, 24, 48 and 72 hours after test substance installation. Irritation of the conjunctivae, which consisted of redness, chemosis and discharge (mean score per animal: 0.7) were observed. The slight irritation had completely resolved within 72 hours in all animals. No iridial irritation or corneal opacity and no corneal epithelial damage was observed. There was no evidence of ocular corrosion.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No irritating effects were observed in in vitro skin and eye irritation test systems. A mild irritancy in eyes was observed in rabbits, but showing complete reversibility and not fulfilling the criteria for classification. As a result, the substance is not considered to be classified for irritancy/corrosivity to the skin and eye under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.