[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Endpoint summary

Description of key information

In vitro tests of the substance in reconstructed human skin models for dermal corrosion and irritation did not result in any indication of adverse effects. However observations in the dermal toxicity study demonstrate that the substance has some potential for inducing dermal irritation, these were not sufficient for classification according to the criteria set out in 1272/2008/EC. With respect to ocular irritation, the BCOP assay proved inconclusive, while the existing in vivo study conducted in rabbits demonstrated only limited and transient conjunctival irritation which resolved by 24 hours; this was not sufficient to classify the substance for ocular irritation according to the criteria set out in 1272/2008/EC. No respiratory irritaiton study has been conducted.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

Incorrect project number assigned to study- This deviation was considered not to affect the purpose or integrity of the study as the required study type was performed on each Test Item

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: OK (640-2) (Furoxy Hydroxy)
CAS number: 56271-94-4
Batch: G316533
Purity: 94.032% w/w
Physical state/Appearance: Cream colored solid
Expiry Date: 25 March 2018
Storage Conditions: Approximately 4 oC in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

other:

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Mattrek EpiDerm
- Tissue batch number(s): 25848
- Shipping date:
- Delivery date: 10 -10 2017
- Date of initiation of testing: 11-10-2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37C
- Temperature of post-treatment incubation (if applicable): 37C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was
achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s
Phosphate Buffered Saline (DPBS) to gently remove any residual test item.
- Observable damage in the tissue due to washing: NO
- Modifications to validated SOP: NO

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hrs
- Spectrophotometer: Labtech LT-4500 microplate reader.
- Wavelength: 570nm
- Filter: not given
- Filter bandwidth: not given
- Linear OD range of spectrophotometer: not given

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The results of the assay are considered acceptable if the following assay acceptance criteria
are achieved:
Negative Control
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of
tissue viability obtained in the testing laboratory after the shipping and storing procedure and
under specific conditions of the assay. The mean OD570 of the two negative control tissues
should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability
meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the
acceptance criterion if mean relative tissue viability of the 60-Minute positive control is
< 15%.
Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates
should be ≤ 30%.
NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 2
- Method of calculation used: mean and standard deviation

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

3 minuts and 60 minutes

Number of replicates:

2 (two)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

ca. 104.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

ca. 103.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

Due to a failure to meet the positive control assay acceptance criteria in the first run it was necessary to repeat the test.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: OK (640-2) (Furoxy Hydroxy)
CAS number: 56271-94-4
Batch: G316533
Purity: 94.032% w/w
Physical state/Appearance: Cream colored solid
Expiry Date: 25 March 2018
Storage Conditions: Approximately 4
The storage temperature did not exceed 8
o
o
C in the dark
C as instructed by the Compound Release
Document.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 3.1 EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 12 December 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-050
Maintenance Medium lot number : 17-MAIN3-055
Assay Medium lot number : 17-ESSC-049

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37C
- Temperature of post-treatment incubation (if applicable): 37C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by
filling and emptying each tissue insert for approximately 40 seconds using a constant soft
stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: N/A

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3mg/mL
- Incubation time: 3hrs
- Spectrophotometer: Labtech LT-4500 microplate reader.
- Wavelength: 570nm
- Filter: N/A
- Filter bandwidth: N/A
- Linear OD range of spectrophotometer: N/A

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
-The results of the assay are considered acceptable if the following assay acceptance criteria
are achieved:
Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue
viability for the positive control treated tissues is ≤40% relative to the negative control treated
tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD570
for the
negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability
is ≤18%.
Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation
calculated from individual percentage tissue viabilities of the three identically treated tissues
is ≤18%.
NUMBER OF REPLICATE TISSUES: 3


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Classification of irritation potential is based upon relative mean tissue viability following the
15-Minute exposure period followed by the 42-Hour post-exposure incubation period
according to the following: relative mean tissue viability <=50% : irritant; relative mean tissue viability>50% Not classified
-

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 (three)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 99.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 4.7% relative to
the negative control treated tissues and the standard deviation value of the viability was 1.8%.
The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.741 and the standard deviation
value of the viability was 8.4%. The negative control acceptance criteria were therefore
satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test
item treated tissues was 1.9%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: OK (640-2) (Furoxy Hydroxy)
Batch: G316533
Purity: 94.032% w/w
Physical state/Appearance: cream colored solid
Expiry Date: 25 March 2018
Storage Conditions: ~4 °C in the dark

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

3.3.1 Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a
by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee
after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with
antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported
to the test facility over ice packs on the same day of slaughter. The corneas were prepared
immediately on arrival.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75mL
- Concentration (if solution): 20%w/v suspension in vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75mL
- Concentration (if solution): 0.9% w/v sodium chloride
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

nil

Number of animals or in vitro replicates:

3 (three)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of
damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to
facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneaswere immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s
Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were
incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer
(Annex 1). The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also
allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
Three per tst item and controls
NEGATIVE CONTROL USED
0.9% w/v sodium chloride
SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED
20% w/v imidazole in 0.9% w/v sodium chloride

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of a 20% w/v solution of the substance in 0.9% w/v sodium chloride

TREATMENT METHOD: [closed chamber / open chamber]
closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD492)
- visual observation

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as per the Test Guideline

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. 8.3

Vehicle controls validity:

valid

Remarks:

IVIS= 1.4

Positive controls validity:

valid

Remarks:

IVIS= 110.8

Remarks on result:

not determinable

Other effects / acceptance of results:

For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the
positive control produced an In Vitro Irritancy Score which fell within two standard
eviations of the historical mean during 2016 for this testing facility. Therefore the In Vitro
Irritancy Score should fall within the range of 65.1 to 123.3.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable
if the negative control produced an In Vitro Irritancy Score which is less than or equal to the
upper limit for background opacity and permeability values during 2016 for bovine corneas
treated with the respective negative control. When testing solids the negative control limit
for opacity should be ≤2.4 and for permeability ≤0.072.

Interpretation of results:

GHS criteria not met

Conclusions:

Not requiring classification for severe eye damage Category 1 to UN GHS or EU CLP. However the result is equivocal for eye irritation category 2/no classification and further infoormaiton is required.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

IN accordance with standard draize methodology but conducted before OECD test guidelines and GLP were introduced

GLP compliance:

no

Remarks:

Study conducted before GLP was introduced

Specific details on test material used for the study:

none given

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Albino rabbits of approximatley 2 kg were used.
No other information on husbandry or environmental conditions is available.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

100mg

Duration of treatment / exposure:

1 hr

Observation period (in vivo):

7 days or as long as injury persists

Number of animals or in vitro replicates:

a minimum of three animals

Details on study design:

The day before the application of test material the eyes are stained with one
drop of a 5% aqueous solution of sodium fluorescein. The stain
is washed out of the eye after 10 seconds and the cornea is then
examined for existing lesions. Any rabbit showing an abnormality
is discarded.
The test material {l00mg or 0.lml) is instilled into the
lower lid of one eye, and then both lids are held together for a
few seconds. The rabbit is restrained in stocks for the next hour.
The eyes are examined and the degree of irritation assessed
1, 24 and 72 hours, and 7 days after application, or as long as
injury persists, by comparison of the test eye with the control
untreated eye. Fluorescein is applied after land 24 hours, but
at subsequent examinations only when an opacity was present on
the previous occasion. The grading of the irritation is based on
modifications of the method of Draize et al (!) and takes account
of the degree of oedema and redness of the bulbar and palpebral
conjunctivae, the quantity of any discharge, the reaction of the
iris to a light source, and the density and area of any opacity of
the cornea. Each test compound is administered to a minimum of
three rabbits.

Irritation parameter:

other: qualitative description

Basis:

other: qualitative description

Time point:

other: maximum of 7 days

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The conclusion of the study was negligible irritation, having effects on the conjunctiva only.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In vitro tests of the substance in reconstructed human skin models for dermal corrosion and irritation did not result in any indication of adverse effects. However observations in the dermal toxicity study demonstrate that the substance has some potential for inducing dermal irritation, these were not sufficient for classification according to the criteria set out in 1272/2008/EC. With respect to ocular irritation, the BCOP assay proved inconclusive, while the existing in vivo study conducted in rabbits demonstrated only limited and transient conjunctival irritation which resolved by 24 hours; this was not sufficient to classify the substance for ocular irritation according to the criteria set out in 1272/2008/EC. No respiratory irritaiton study has been conducted.