.alpha.-D-Glucopyranoside, methyl 1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, compd. with 2-butyne-1,4-diol (1:1)

Endpoint summary

Description of key information

A key skin irritation in-vivo study in rabbits is available.  For eye irritation classification two studies are available; an in-vitro BCOP study  followed by aan in-vivo sentinel one rabbit study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 15- March 2, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

New Zealand White rabbits were supplied by David Percival Ltd, Moston, Sandbach, Cheshire, UK.; acclimatisation period of at least five days; At the start of the study the animals were in the weight range of 2.0 to 3.5 kg and
were twelve to twenty weeks old. Free access to mains drinking water and food was allowed through out the study. The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70% respectively. The rate of air exchange was at least 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: distilled water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 g of the test material, moistened with 0.5 ml of distilled water was introduced under 2.5 cm X 2.5 cm cotton gauze patch

Duration of treatment / exposure:

One animal was intially treated at three sites. One patch was removed at each of these 3 times: 1 minute, one hour and 4 hours after application. The patch was removed at the indicated times and any residual material removed from the test area by gentle swabbing with cotton wool soaked in distilled water. After review of the first animal data, 2 additional animlas were treated where one patch was applied and allowed to remain for 4 hours after application.

Observation period:

Approximately one hour after removing the patch and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored.

Number of animals:

3

Details on study design:

The day before the test the rabbits were clipped free of fur from the dorsal/flank area and inspected for gross abnormalities of the epidermis. Only animals with an intact healthy epidermis are selected for the study. A suitable site on the back of the rabbit was selected as test site and 0.5 g of test material moistened with 0.5 ml of distilled water was introduced under a 2.5 cm X 2.5 cm cotton gauze patch which was secured in position with surgical tape. For animal one there were three patches which were removed 3 mintes, 1 hour and 4 hours after application. After the patchwas removed, the test sites were evaluated after 1 hour, 24, 48, and 72 hours according to the Draize classification scheme for determination of primary irritation index.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 24hr 72hr

Score:

1.5

Reversibility:

fully reversible

Remarks on result:

other: mild irritant

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

The individual scores for erythema/eschar and oedema were very slight and never above a 1 for all three animals for the complete 72 hour observation period. The Draize classification scheme for determination of primary irritation index was used. Results were fully reversible.

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize

Scheme: The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test

rabbits (12 values) and this total was divided by six to give the primary irritation index of the test

material.

Primary Irritation Index Classification of Irritancy

0                                   Non-irritant

>0 to2                            Mild irritant

>2 to 5                            Moderate irritant

> 5 to 8                           Severe irritant

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material produced a primary irritation index of 1.5 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. The test material did not meet the classification as irritant or corrosive according to the EU labelling regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July, 2015 - 19 August, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

(2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

(1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: the animal was 18 weeks old
- Weight at study initiation: 3291 grams
- 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 set to maintain
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

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 applied: 19.6 mg (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eye of the animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance.

Number of animals or in vitro replicates:

1 male

Details on study design:

WEIGHT OF EVIDENCE ANALYSIS
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed, prior to the start of this in vivo eye irritation study in the rabbit. As recommended in the test guidelines, all available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity) and in vitro, ex vivo and in vivo tests) to determine the need for in vivo eye testing.
Based on the available information, it was concluded that there was the need to perform this in vivo eye irritation study in rabbit in order to establish the possible eye irritating properties of the test substance.

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). Based on the duration of the ocular lesions, the test substance could be classified without treating the two further rabbits assigned to the study.

Preemptive pain management:
One hour prior to instillation of the test substance, buprenorphine 0.01 mg/kg bw was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5% were applied to both eyes.

TREATMENT
The animal was treated by instillation of 19.6 mg of the test substance (a volume of approximately 0.1 mL), 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 water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.03 mg/kg bw and meloxicam 0.5 mg/kg bw were administered by subcutaneous injection.
Additional injections of buprenorphine 0.03 mg/kg bw were supplied on Day 2 and after the 48-hour observation (buprenorphine 0.01 mg/kg bw and meloxicam 0.5 mg/kg bw) to reduce pain and distress.
After the final observation, the animal was euthanatised by intra-venous injection of Euthasol® 20%.

REMOVAL OF TEST SUBSTANCE
-Washing (if done): No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation: The eyes of the animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days 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.

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

animal #1

Remarks:

(mean)

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

(mean)

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

(mean)

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

(mean)

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritant / corrosive response data:

Instillation of 19.6 mg of BMS-587319-03 (a volume of approximately 0.1 mL) into an eye of one rabbit resulted in severe effects on the cornea, iris and conjunctivae which did not completely resolve within 21 days.
The corneal injury consisted of opacity and epithelial damage. Iridial irritation (max. Grade 2) was observed and the irritation of the conjunctivae consisted of redness (max. grade 3), chemosis (max. grade 3) and discharge (max. grade 2).
There was no evidence of ocular corrosion.

Other effects:

No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.
No mortality occurred and no signs of systemic toxicity were observed in the animal during the test period.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an eye irritation study with a male rabbit, performed according to OECD 405 test guideline and GLP principles, moderate to severe irritation was observed, which was not fully reversible within 21 days (duration of the study).

Executive summary:

BMS-587319-03 was tested in an eye irritation study with a male rabbit, performed according to OECD 405 test guideline and GLP principles.

Instillation of BMS-587319-03 into an eye of one rabbit resulted in severe effects on the cornea, iris and conjunctivae which did not completely recover within 21 days. The corneal injury consisted of opacity and epithelial damage. Iridial irritation (max. Grade 2) was observed and the irritation of the conjunctivae consisted of redness (max. grade 3), chemosis (max. grade 3) and discharge (max. grade 2). There was no evidence of ocular corrosion.

Based on the results, BMS-587319-03 should be classified as Irreversible effects on the eye (Category 1) and labeled with H318: Causes serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data on acute inhalation irritation is lacking, however the substance is classified for severe eye damage 1 by the in vivo rabbit assay. Based upon the particle size distribution and the corrosive properties of 2-butyne-1,4-diol, there is potential for upper respiratory tract irritation.

Justification for selection of skin irritation / corrosion endpoint:
Mildly and/or transiently irritating to skin.

Justification for selection of eye irritation endpoint:
The in vitro BCOP assay was inconclusive with an in vitro score of 51.8 indicating potential irritation. Further testing of the substance in a single sentinel animal resulted in irreversible eye damage at 21 days.

Effects on skin irritation/corrosion: No adverse effect observed (not irritating)

Effects on eye irritation: corrosive

Justification for classification or non-classification

The test material produced a primary irritation index of 1.5 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. The test material did not meet the classification as skin irritant or corrosive according to the EU labelling regulations.

The result of testing of BMS 587319-03 in the BCOP assay was inconclusive with an in vitro score of 51.8 indicating potential irritation. Further testing of the substance in a single sentinel animal resulted in irreversible eye damage at 21 days; consequently, it is classified as Serious Eye Damage Category 1 in accordance with the criteria set out in CLP.

Based upon the harmonised classification for but-2 -yne-1,4 -diol (EC 603 -076 -00 -9) of Skin corrosion (H314) and the particle distribution profile where >70% of the particles are inhalable, there is significant potential for respiratory irritation. Consequently, classification for Respiratory Irritation (STOT-SE H335) is justified.