Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06Feb2015 to 10 Mar2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

EC Number:

269-503-2

EC Name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Cas Number:

68259-00-7

Molecular formula:

C14H16N3.CH3O4S

IUPAC Name:

methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Test material form:

solid: particulate/powder

Details on test material:

See study reports for batch and purity information

Specific details on test material used for the study:

Test material name : Vibracolor® Citrus Yellow
Chemical name : Methyl -1-methyl-4-((methylphenylhydrazono)
methyl)pyridinium sulphate
Batch number : 0010241302
BASF Test Item no.
(Container identification) : 10/0384-4
Purity : 99.2% (see Annex 3)
Appearance : solid, yellow
Storage conditions : ambient temperature (15-25ºC)
Expiry date : 21/06/2018

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

Characterization of the test system
Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5 - 2.5 kg, were used as eye-donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Experimental design
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If ndamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.

The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO Triskelion, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10 - 0.15 mL/min (peristaltic pump set at speed 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32oC (water pump set at 36.4oC; Lauda 103, Germany).

After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.

Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

Test system

Vehicle:

other: Either directly (30 mg) or as 5% aqueous solution (30 μL)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

Negative control; Physiological saline; 30 μL
Positive control; NaOH; 30 mg
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 30 mg
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 30 μL

Duration of treatment / exposure:

Negative control; Physiological saline; 10 seconds exposure then washed with 20mL saline
Positive control; NaOH; 10 seconds exposure then washed with 20mL saline
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 10 seconds exposure then washed with 20mL saline
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 10 seconds exposure then washed with 20mL saline

Duration of post- treatment incubation (in vitro):

After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

Negative control; Physiological saline; 1 eye
Positive control; NaOH; 3 eyes
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 3 eyes
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 3 eyes

Details on study design:

Negative control; Physiological saline; 10 seconds exposure then washed with 20mL saline
Positive control; NaOH; 10 seconds exposure then washed with 20mL saline
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 10 seconds exposure then washed with 20mL saline
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 10 seconds exposure then washed with 20mL saline

After rinsing, each eye in the holder was returned to its chamber.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment.
Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Vibracolor Citrus Yellow 30 mg

Value:

4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Vibracolor Citrus Yellow 30 mg

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Vibracolor Citrus Yellow 30 mg

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Slit-lamp examination
An overall summary of the results of the control (negative and positive) and test substances based on maximum mean values for corneal swelling, corneal opacity, and fluorescein retention, the irritation categories assigned to the parameters. The individual and mean values for corneal swelling, corneal opacity and fluorescein retention are recorded in the study records.

Vibracolor® Citrus Yellow caused corneal effects consisting of very slight corneal swelling (mean of 4%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). The opacity and fluorescein retention were observed as spots. In addition, yellow staining of cornea and sclera was observed which persisted until the end of the observation period.

The 5% aqueous dilution of Vibracolor® Citrus Yellow only caused very slight corneal swelling (mean of 1%). The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate

The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination
Since the test sample concerned a dye substance, the vitreous body of each eye was examined for possible staining during collection of the corneas in formalin and no staining was observed.

Microscopic examination of the corneas treated with Vibracolor® Citrus Yellow revealed very slight erosion of the epithelium in two corneas. Microscopic examination of the corneas treated with a 5% aqueous dilution of Vibracolor® Citrus Yellow did not reveal any abnormalities.

Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, pyknotic nuclei in the inner region of the stroma and necrosis of the endothelium.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Applying the classification criteria of the ICE, the following irritation classifications can be assigned to the test item:
- Category 2A:“Irritant/causes eye irritation” (UN-GHS classification);
- Category 2:“Irritating to eyes” (EU-CLP classification).

5% aqueous dilution of the test item:
- Not Classified (UN-GHS and EU-CLP classifications).

Executive summary:

Test item and a 5% aqueous dilution of the test item were evaluated for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 μL (liquids) or 30 mg (solids) for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

 

Vibracolor® Citrus Yellow caused corneal effects consisting of very slight corneal swelling (mean of 4%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). The opacity and fluorescein retention were observed as spots. In addition, yellow staining of cornea and sclera was observed which persisted until the end of the observation period. The 5% aqueous dilution of the test item only caused very slight corneal swelling (mean of 1%).

 

Since the test sample concerned a dye substance, the vitreous body of each eye was examined for possible staining during collection of the corneas in formalin and no staining was observed. Microscopic examination of the corneas treated with the test item revealed very slight erosion of the epithelium in two corneas. Microscopic examination of the corneas treated with a 5% aqueous dilution of the test item did not reveal any abnormalities.

 

The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, pyknotic nuclei in the inner region of the stroma and necrosis of the endothelium.

 

Applying the classification criteria of the ICE, the following irritation classifications can be assigned to the test item:

-Category 2A:“Irritant/causes eye irritation” (UN-GHS classification);

-Category 2:“Irritating to eyes” (EU-CLP classification).

5% aqueous dilution of the test item:

-Not Classified (UN-GHS and EU-CLP classifications).