Dimethyl (p-methoxybenzylidene)malonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-06 to 2017-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl (p-methoxybenzylidene)malonate

EC Number:

231-185-8

EC Name:

Dimethyl (p-methoxybenzylidene)malonate

Cas Number:

7443-25-6

Molecular formula:

C13H14O5

IUPAC Name:

1,3-dimethyl 2-[(4-methoxyphenyl)methylidene]propanedioate

Test material form:

solid

Specific details on test material used for the study:

Name: Hostavin PR-25 P
Chemical Name: Propanedioic acid, [(4-methoxy-phenyl)-methylene]-,dimethyl ester
Product (Common Name/Code): Hostavin PR-25 P
CAS No.: 7443-25-6
Batch No.: CHA0122531
Molecular Weight: 250 g/mol
Physical State: solid
Colour: white
Density: 1.348
Purity: 99.8%
Date of Analysis: 27 January 2017
Expiry Date: 15 March 2018
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test System
Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: female
Body weight at the
beginning of the study: animal no. 1: 4.8 kg
animal no. 2: 4.6 kg
Age at the
beginning of the study: animal no. 1: approximately 29 weeks old
animal no. 2: approximately 31 weeks old
Number of animals: 2
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes. 
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Semi barrier in an air-conditioned room
- Temperature: 18 +/- 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A dose of 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball.
The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The treated eye of animal no. 1 was not rinsed after the application. The treated eye of animal no. 2 was rinsed with physiological saline 0.9% NaCl 1 hour after the application due to residues of the test item at the animals eye.

Observation period (in vivo):

The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period was extended up to 8 days after dosing.

Number of animals or in vitro replicates:

The in vivo test was performed initially using one animal.
The results of the initial test did not indicate the test item to be corrosive but a severe irritant to the eye using the procedure described.
In order to confirm the response, one additional animal was treated in the same manner.
According to OECD 405, section 23, treatment of a third animal was omitted as results from the second animal were sufficient to allow for hazard classification determination.

Details on study design:

Preparation of the Animals
Within 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was performed to ensure the good state of health of the animals.
Approximately 16 hours (animal no. 1) and 15 hours (animal no. 2) before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo, Alcon Pharma, lot no. H 603, expiry date: 08/2018). The eyes were rinsed with physiological saline 0.9% NaCl (Alleman Pharma, lot no. 609709, expiry date: 08/2019) after the examination.
None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Application
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no.: 5481, expiry date: 01/2018 (animal no. 1) and lot no.: 5510, expiry date: 09/2018 (animal no. 2)) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia.
Approximately 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (proparacaine hydrochloride ophthalmic 0.5% solution, URSAPHARM, lot no.: 286171, expiry date: 07/2017) were administered in both the treated and the control eye of each animal.
The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The treated eye of animal no. 1 was not rinsed after the application. The treated eye of animal no. 2 was rinsed with physiological saline 0.9% NaCl 1 hour after the application due to residues of the test item at the animals eye.
To prevent pain and distress after the application of the test item both animals were treated with the following doses of buprenorphine and meloxicam:

Clinical Observation
The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according to the Grading System for Ocular Lesion.
For the calculation only the 24, 48 and 72-hour readings were used.
72 hours post-application as well as at the end of the prolonged observation period the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

Body Weight
The animals were weighed prior to the administration and at the end of the observation period.

Evaluation of Results
The animal’s reactions were recorded according to the scoring system described at each time of observation.
For the calculation only the 24, 48 and 72-hour readings were used.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 8 days

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 8 days

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 8 days

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

After the application into the eyes of two female NZW rabbits the test item produced irritant but not corrosive ocular effects.
Neither mortality nor significant clinical signs of toxicity but local effects were observed.
At the end of the prolonged observation period no corneal lesions were found.
Conjunctival redness, chemosis and hypersecretion were observed and are described in the report.
Clinical findings and local changes (hypersecretion with moistening of the lids and surrounding hair, hypersecretion with wetting of the eyelids and hair around the eyes and clear flow secretion) were observed.

Other effects:

The body weight development of animal no. 1 was within the expected range. For animal no. 2 a moderate weight loss due to reduced food and water intake was observed

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of the present study, a single ocular application of the test item Hostavin PR-25 P to rabbits at a dose of 0.1 g produced irritant effects (Chemosis > 2), which were fully reversible within 8 days.
Neither mortalities nor significant clinical signs of toxicity but local effects were observed.

Executive summary:

Summary Results

Species/strain: New Zealand White Rabbits Crl: KBL (NZW)

Number of animals: 2

Amount of substance: 0.1 g per test site

First time of effects: animal no. 1: 1 hour post-application redness grade 2, chemosis grade 3, 24 hours post-apllication hypersecretion grade 1

animal no. 2: 1 hour post-application redness grade 1, chemosis grade 2

Last time of effects: animal no. 1: 48 hours post-application hypersectretion grade 1, 72 hours post-application chemosis grade 1 and 7 days post-application redness grade 1

animal no. 2: 5 days post-application chemosis grade 1 and 6 days post-application redness grade 1

Reversibility of the observed effects:  the changes were fully reversible within 8 days post-application

Method: OECD 405, EC 440/2008, Method B.5, OPPTS 870.2400

Conclusion

Under the conditions of the present study, a single ocular application of the test item Hostavin PR-25 P to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 8 days. Neither mortalities nor significant clinical signs of toxicity but local effects were observed.