Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-18 till 2009-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

EC Number:

266-273-5

EC Name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Cas Number:

66241-11-0

Molecular formula:

Molecular formula is not available.

IUPAC Name:

Reaction products of 2,4-dinitrophenol with polysulfide, leuco derivatives, sodium salt

Details on test material:

- Name of test material : Leuco Sulfur Black 1
- Physical state: thixotrophic, black, wet paste
- Composition: Leuco Sulfur Black 1:52.75 % (w/w) (calculated by amount of by-compounds)
2,4-Dinitrochlorobenzene: < 20 ppm
2,4-Dinitrophenole: < 20 ppm
Sulfate: 970 ppm
Chloride: 750 ppm
Sodium: 1.3 % (w/w)
Water: 45.75 % (w/w) by Karl-Fischer titration
- Lot/batch No.: ESA0007595
- Expiration date of the lot/batch: 2011-07-11
- Stability under test conditions: Stable under storage conditions.
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS: Young Adult New Zealand White Rabbit, SPF
- Source: Harlan Laboratories B.V. 5960 AD Horst / The Netherlands
- Age at study initiation: 15 weeks (male), 15 - 16 weeks (females)
- Weight at study initiation: 2608 g (male), 2729 - 2892 g (females)
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks and haysticks for gnawing.
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum
- Acclimation : Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study
- Randomization: Selected by hand at time of delivery. No computer generated randomization program.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): between 30-70% (values above 70% during cleaning process possible)
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: left eye of each animal treated, right eye served for reference control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1g/ animal (undiluted)



VEHICLE: none

Duration of treatment / exposure:

The eyes of all animals were rinsed with sterile physiological saline (B. Braun, Emmenbrücke, Switzerland; Lot-No. 7443A191, expiry date 30-Sep-2009) after the 1-hour observation because black test item remnants (particles) were observed at this observation time point.

Observation period (in vivo):

72h

Number of animals or in vitro replicates:

Number of Animals per Test: 3 (Animals of both sexes were used)

Details on study design:

REMOVAL OF TEST SUBSTANCE
The eyes of all animals were rinsed with sterile physiological saline (B. Braun, Emmenbrücke, Switzerland; Lot-No. 7443A191, expiry date 30-Sep-2009) after the 1-hour observation because black test item remnants (particles) were observed at this observation time point.


SCORING SYSTEM:
The eye reactions were assessed according to the numerical scoring system listed in the
Commission Regulation (EC) No. 440/2008, B.5, at approximately 1, 24, 48 and 72 hours after
administration. Scleral reddening and ocular discharge were also assessed.

TOOL USED TO ASSESS SCORE:
Eye examinations were made with a Varta Cliptrix diagnostic-lamp

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 24 hours

Score:

0

Max. score:

0

Results 2

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 48 hours

Score:

0

Max. score:

0

Results 3

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 72 hours

Score:

0

Max. score:

0

Irritant / corrosive response data:

no irritation effects observed

Other effects:

Viability / Mortality: No mortality occurred.
Clinical Signs: No clinical signs were observed during the course of the study.
Body Weights: The body weights of all rabbits were considered to be within the normal range of variability.
Pathology, Macroscopic Findings: No necropsy was performed at the end of the study.

Any other information on results incl. tables

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for all three animals for the conjunctival reddening and swelling were 0.00 and 0.00, respectively.

No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals. Slight conjunctival reddening and swelling, slight scleral reddening, as well as slight ocular discharge was noted in all three animals at the 1-hour reading.

No abnormal findings were observed in the treated eye of any animal 72 hours after treatment, the end of the observation period for all animals.

Coloration: No staining produced by the test item of the treated eye was observed.

Test Item Remnants: Black test item remnants were evident in the eye or conjunctival sac of all three animals at the 1- hour reading.

Corrosion: No corrosion of the cornea was observed at any of the reading times.

Eye Irritation Scores

Animal Number	Sex	Evaluation Interval*	Corneal Opacity	Area of Corneal Opacity	Iris	Conjunctivae
						Redness	Chemosis	Sclera
19	M	1 hour	0	0	0	1	1	1
20	F		0	0	0	1	1	1
21	F		0	0	0	1	1	1
19	M	24 hours	0	0	0	0	0	0
20	F		0	0	0	0	0	0
21	F		0	0	0	0	0	0
19	M	48 hours	0	0	0	0	0	0
20	F		0	0	0	0	0	0
21	F		0	0	0	0	0	0
19	M	72 hours	0	0	0	0	0	0
20	F		0	0	0	0	0	0
21	F		0	0	0	0	0	0

* Examinations were performed at the specified times after instillation of the test item.

Eye Irritation Scores – Mean Values after 24, 48 and 72 Hours

Animal Number	Sex	Corneal Opacity	N	Iris	N	Conjunctivae
						Redness	N	Chemosis	N
19	M	0.00	3	0.00	3	0.00	3	0.00	3
20	F	0.00	3	0.00	3	0.00	3	0.00	3
21	F	0.00	3	0.00	3	0.00	3	0.00	3

N = number of available data points

Eye Irritation Scores – Assessment According to EC Guidelines

Evaluated intervals	Corneal Opacity	Iris	Conjunctivae
			Redness	Chemosis
24 hours	Not Irritating	Not Irritating	Not Irritating	Not Irritating
48 hours
72 hours

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based upon the referred classification (Commission Directive 2001/59/EC of August 06, 2001), Leuco Sulfur Black 1 is considered to be “not irritating” to the rabbit eye.

Executive summary:

The primary eye irritation potential of Leuco Sulfur Black 1 was investigated according to OECD test guideline No. 405 and Commission Regulation (EC) No. 440/2008, B.5. The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for all three animals for the conjunctival reddening and swelling were 0.00 and 0.00, respectively.

The instillation of Leuco Sulfur Black 1 into the eye resulted in mild, early-onset and transient ocular changes, such as reddening and swelling of the conjunctivae, redness of the sclera as well as discharge. These effects were reversible and were no longer evident 24 hours after treatment in all animals. Black test item remnants were observed in the eye or conjunctival sac of all three animals at the 1-hour reading. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals.

No clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), Leuco Sulfur Black 1 is considered to be “not irritating” to the rabbit eye.