2-[(2S,3S)-3-[[(1,1dimethylethoxy)carbonyl]amino]-2-hydroxy-4-phenylbutyl]-2-[[4-(2pyridinyl)phenyl]methyl]-,1,1-dimethylethyl ester

Endpoint summary

Description of key information

Skin irritation potential of the substance BMS 233101-01 was tested according to EU Test Method B4, Directive 92/69/EEC and OECD Guideline 404. 500 mg of the test substance were diluted in distilled water and applied semi-occlusively on 3 New Zealand White rabbits. The test material produced a primary irritation index of 0.0 (according to the Draize classification scheme) and no corrosive effects were noted. Therefore, it does not meet the criteria for classification as irritant or corrosive according to the CLP Regulation.

The test substance was found to be mildly and/or transiently irritating to eyes with a IVIS value of <3 in a BCOP study however this was insufficient for classification. Potential of the substance BMS 233101-01 for causing eye damage or irritation was further tested according to EU Test Method B5, Directive 92/69/EEC and OECD Guideline 405. The test substance was applied to 3 New Zealand White rabbits. Mild, transient irritation was observed. Iridial inflammation was noted in one treated eye at the 1-hour observation. No other iridial effects were noted. Moderate conjunctival irritation was noted at the 1-hour observation with minimal conjunctival irritation apparent at the 24-hour observation. All treated eyes appeared normal at the 48-hour observation. No corneal alterations were observed. As a result, the substance is not classified for causing eye irritation or eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

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:

key study

Study period:

February 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted prior to changes in Annex VI requirements

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

Study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit.

GLP compliance:

yes

Specific details on test material used for the study:

BMS233101, purity 99.95%, off white solid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Three New Zealand White rabbits, supplied by David Percival Ltd, Moston, Sandbach, Cheshire, UK, were used. At the start of the study the animals weighed 2.79 to 3.15 kg and were twelve to sixteen weeks old. After a minimum acclimatisation period of five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended metal cages. Free access to mains drinking water and food {STANRAB SQC Rabbit Diet, Special Diets
Services Ltd, Witham, Essex, UK) was allowed throughout the study.
The animal room was maintained at a temperature of 17 to 21 'C and relative humidity of 48 to 65%. The rate of air exchange was approximately fifteen
changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml of the test material, which was found to weigh approximately 49 mg

Observation period (in vivo):

1 hour and 24, 48 and 72 hours following treatment

Number of animals or in vitro replicates:

3 animals

Details on study design:

One rabbit was initially treated. A volume of 0.1 ml of the test material, which was found to weigh approximately 49 mg {as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctiva! sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hrs.

Irritant / corrosive response data:

No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye at the 1-hour observation. No other iridial effects were noted.
Moderate conjunctival irritation was noted at the 1-hour observation with minimal conjunctival irritation apparent at the 24-hour observation. All treated eyes appeared normal at the 48-hour observati

Interpretation of results:

GHS criteria not met

Conclusions:

The test material, BMS 233101-01, produced a maximum group mean score of 11.7 and was classified as a MINIMAL IRRITANT (CLASS 3 ON A 1 TO 8 SCALE) to the rabbit eye according to a modified Kay and Calandra classification system.
The test material did not produce positive criteria in any rabbit according to the EU labelling regulations. No symbol and risk phrase are therefore required.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-10-20 to 1998-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

- Principle of test: The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the ocular irritancy of the test article to isolated bovine corneas
- Short description of test conditions:Bovine corneas, obtained as a by-product from freshly slaughtered animals, were mounted in special holders and exposed to the test article
- Parameters analysed / observed: An in vitro score was determined for the test article based on the induction of opacity and permeability to fluorescein in the isolated bovine corneas.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 98AF95

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted in saline (0.9%, USP; Baxter)

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Test article in saline (0.9%, USP; Baxter) to achieve a 20% mixture

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Byproduct from a local abattoir
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised and then placed in Hanks' Balanced Salt Solution, suplemented with Penicillin/Streptomycin and transported to the laboratory on ice packs. They were used within 24 hours of receipt.
- Time interval prior to initiating testing: 24 hours
- indication of any existing defects or lesions in ocular tissue samples: All eyes with existing defects/damage were removed prior to the start of the study.
- Indication of any antibiotics used: Yes, penicillin/streptomycin

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 ul of solution was applied to each eye
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 ul of solution was applied to each eye
- Concentration (if solution): 80%

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

2 for each control, and 3 for the test solution

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then positioned on top of the cornea and the screws were tightened. Starting with the posterior compartment, the two compartments of the corneal holder were then filled with Minimum Essential Medium Eagle (MEM) without phenol red, with 1 % fetal bovine serum (complete MEM). The corneal holders were incubated at 32 ± 10C for a minimum of one hour.
QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were grossly examined for damage and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea
NUMBER OF REPLICATES
2 for each control, and 3 for the test solution
SOLVENT CONTROL USED (if applicable)
Yes
POSITIVE CONTROL USED
Yes, imidazole
APPLICATION DOSE AND EXPOSURE TIME
750 ul of test solution and 4 hours
TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: yes, 60 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2 washing steps, with at least 3 washings with complete MEM the first step and 1 washing with complete MEM for the second step.
- POST-EXPOSURE INCUBATION:
Yes, 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer at study intiation and completion
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a kinetic microplate reader(OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Score = Mean Opacity Value + (15 x Mean 00490 Value)

DECISION CRITERIA: The BCOP assay was accepted when the positive control, imidazole, caused an in vitro score that fell within two standard deviations of the historical mean.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Solution

Value:

ca. 2.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The results of the positive control fell within two standard deviations of the historical mean (within a range of 78.6 to 143.9), the assay was considered valid.

Interpretation of results:

GHS criteria not met

Conclusions:

This study validly found that the test substance is not irritating to the eyes according to GHS criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The test material produced a primary irritation index of 0.0 (according to the Draize classification scheme) and no corrosive effects were noted. Therefore, it does not meet the criteria for classification as irritant or corrosive according to the CLP Regulation.

Mild, transient irritation was observed, but it was not sufficient for classification according to CLP.