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Administrative data

Description of key information

Data in vitro and in vivo indicate that the substance is not a skin or eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 October 2011 to 5 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In vitro assay - EPISKIN - performed in accordance with Guideline OECD 439 with no remarkable deviations or deficiencies
Qualifier:
according to
Guideline:
other: OECD Guideline 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (22 July 2010); Commission Regulation (EC) No. 761/2009 amending Regulation (EC) No. 440/2008, B.46 In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test
Principles of method if other than guideline:
EPISKIN reconstructed human epidermis model
GLP compliance:
yes
Test system:
human skin model
Remarks:
cultured human epidermal keratinocytes
Source species:
other: Human
Cell type:
other: human derived epidermis keratinocytes
Cell source:
other: reconstructed human epidermis (RhE) model
Details on test system:
Test model
- Source: EPISKIN - reconstructed human epidermis - supplied by SkinEthic Laboratories, Nice, France
- Received at laboratory: 29 November 2011
- batch: 11-EKIN-044
- Functional controls: Quality controls: histology scoring, magnitute of viability and barrier function (IC50 determination).
Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.


IN-LIFE DATES: Experimental start date: 18 October 2011; experimental completion date: 5 December 2011
Type of coverage:
other: 0.38 cm2 EPISKIN
Preparation of test site:
other: Test system examined on arrival - pale grey temperature indicator and orange pH indicator meaning te system was suitable for use. Culture dishes were incubated for 24 hours after arrival at 37C, at saturated humidity and 5% CO2.
Vehicle:
other: Various media used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL

Negative control - D-PBS 20uL/well; three replicates

Positive control - 5% SDS in water 20uL/well; three replicates

Test material - 4,4-biphenol 20 mg/well; three replicates

Media:
SkinEthic Maintenance Medium: SkinEthic; batch: 11-MAIN3-060
SkinEthic Assay Medium : SkinEthic; batch: 11-ESSC-037 (Preliminary Phase) and 11-ESSC-048 (Main Assay)
Dulbecco’s Phosphate buffered saline (D-PBS): GIBCO; batch: 925780
Sterile water : Bieffe Medital; batch: 08K2901
MTT Reagent : Sigma; batch: MKBB1495
MTT Stock Solution : MTT Reagent 3 mg/ml in D-PBS
Stored at ambient conditions, protected from light; used on the day of dilution
MTT Ready-to-use Solution : MTT Stock Solution diluted 1:10 (v/v) with SkinEthic Assay Medium (final concentration 0.3 mg/ml of MTT)
Stored at ambient conditions, protected from light. Used within 1 hour from preparation
Acidic Isopropanol (0.04 N HCl in isopropanol): 12 N HCL (Analab; batch: 08B22050j) added to 2-propanol (Fluka; batch: 0001434790)
Duration of treatment / exposure:
In the preliminary test, the non-specific reduction of MTT was evaluated. 2ml of MTT Ready-to-Use solution was incubated with 10 mg of the test material for 3 hours (saturated humidity, 5%CO2 at 37°C). The solution was examined after incubation for blue or purple appearance. The colouring potential interaction with the test system was tested by adding 10 mg of test material to 90 uL distilled water and vortex mixed for 15 minutes. The solution colour was evaluated at the end of the incubation time.

In the main assay, the test material and positive and negative controls were applied to live tissue, three replicates. The exposure period was 15 minutes; after exposure each tissue was rinsed with sterile D-PBS and transferred to a new well with 2 ml maintenance medium. The wells were incubated at saturated humidityand 5%CO2 at 37°C for the 42 hour recovery period. At the end of the recovery period samples of incubation medium were reserved (but not subjected to further analyses). The tissue inserts and controls were then incubated with 2 ml MTT ready to use solution for three hours at 37°C (saturated humidity, 5%CO2) .
Tissues were then removed, dried on absorbent paper and a total biopsy obtained using a biopsy punch.
Observation period:
Not applicable
Number of animals:
Reconstructed human epidermal tissue. Three replicate live tissues per treatment
Details on study design:
In the main assay, the test material and positive and negative controls were applied to live tissue, three replicates. The exposure period was 15 minutes; after exposure each tissue was rinsed with sterile D-PBS and transferred to a new well with 2 ml maintenance medium. The wells were incubated at saturated humidity, 5%CO2 at 37°C for the 42 hour recovery period. At the end of the recovery period samples of incubation medium were reserved (but not subjected to further analyses). The tissue inserts and controls were then incubated with 2 ml MTT ready to use solution for three hours at saturated humidity, 5%CO2 at 37°C.
Tissues were then removed, dried on absorbent paper and a total biopsy obtained using a biopsy punch.

The epidermis was separated from the collagen matrix and both elements were placed into a microtube containing 500 uL of acidic isopropanol. The tubes were vortex mixed and preserved for three days at 4°C to allow for formazan extraction. After extraction and centrifugation to remove debris, 200 µL aliquots were read in duplicate for absorbance at 595 nm. OD values were recorded. Isopropanol samples were used as control blanks.
Irritation / corrosion parameter:
% tissue viability
Value:
119.4
Remarks on result:
no indication of irritation
Remarks:
Mean score from 3 replicates
Interpretation of results:
GHS criteria not met
Conclusions:
The potential of the test item 4,4-Biphenol to be irritant to skin was investigated in an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN. According to the histotoxic potential, when referred to the negative control, the test item is considered to have no effect on the test system (119.4% of mean cell viability when compared to negative control). The negative and positive controls gave the expected results and the study was accepted as valid.

According to the criteria defined in the guidelines for this test (cell viability less than 50%), the test item is not considered to have irritant effect on the skin under the reported experimental conditions.
Executive summary:

The potential for 4,4'-Biphenol to be irritant to the skin was investigated in an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model - EPISKINTM. 4,4'-Biphenol as well as positive and negative controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system as an index of cell viability. A preliminary test was carried out to assay the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and of colouring water per se. No interaction was recorded between the test item and MTT under the normal test condition. No colouring potential was recorded for the test item in contact with water. Therefore, no additional control was added to the main phase for the evaluation of non specific coloration which may have influenced evaluation of results. In the main assay, 4,4'-Biphenol was applied, as supplied, in three replicates at the treatment level of approximately 20 mg/epidermis unit (0.38 cm2 each). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, using the same number of replicates and test conditions, at a dose level of 20 μl/epidermis unit. The negative control gave the expected baseline value and variability, in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death when compared to the negative control (10.1% of cell viability) even with a variability slightly higher than expected (CV% equal to 33.8 instead of less than 18), but the test was considered valid. 4,4'-Biphenol did not induce cell death, the mean viability was 119.4% with reference to the negative control. According to the established criteria (cell viability less than 50%), the test item is considered to have no irritant effect on the skin under the reported experimental conditions.

Endpoint:
skin irritation / corrosion
Remarks:
no data
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
no details provided in publication issued in 1974
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Insufficient details provided for either methods or results
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test material applied uncovered to rabbit skin. No details of skin preparation or method of assessing reactions. A grade of response is quoted without any context for assessing the meaning of the individual grade.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Details on test animals and environmental conditions:
no data
Type of coverage:
open
Preparation of test site:
not specified
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
No details provided
Duration of treatment / exposure:
No details provided
Observation period:
no details provided
Number of animals:
No data
Details on study design:
No details are provided for the methods used in this range-finding screen. According to the tabulated results a rabbit or rabbits was/were exposed to the test material on the belly, without occlusion. Information relating to how or how long the material was retained in situ is not provided. A footnote indicates a suitable vehicle was used but does not give further details on the nature of the vehicle.
Interpretation of results:
study cannot be used for classification
Remarks:
Without a reference point the quoted value of "3" is meaningless
Conclusions:
A valid conclusion cannot be drawn given the sketchy details of the methods and isolated nature of the results score.
Executive summary:

The study does not provide sufficient information on which to base a valid judgement of skin irritancy potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Remarks:
no data
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
No details presented in the publication
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Insufficient details provided for either methods or results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Corneal injury assessed following instillation into rabbit eye or eyes. No further details of method provided. The test material was dispersed in a vehicle prior to instillation. No details of the grading system are provided to indicate how the point value stated can be interpreted.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
No data
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
No details provided
Duration of treatment / exposure:
No details provided
Observation period (in vivo):
No details provided
Number of animals or in vitro replicates:
No details provided
Details on study design:
No details provided. Corneal injury was assessed in one or more rabbits in a screening/range-finding assay.
Irritation parameter:
cornea opacity score
Basis:
animal: no data about the number of tested animals
Time point:
other: no data
Score:
3
Reversibility:
not specified
Remarks on result:
other: A score of 3 is recorded but no grading system to indicate how this value relates to a degree of irritation
Irritation parameter:
iris score
Basis:
animal: no data
Time point:
other: no data
Reversibility:
not specified
Remarks on result:
not measured/tested
Irritation parameter:
conjunctivae score
Basis:
animal: no data
Time point:
other: no data
Reversibility:
not specified
Remarks on result:
not measured/tested
Irritation parameter:
chemosis score
Basis:
animal: no data
Time point:
other: no data
Reversibility:
not specified
Remarks on result:
not measured/tested
Irritant / corrosive response data:
Scored as 3. No indication given in the publication to aid interpretation of the value.
Other effects:
No details
Interpretation of results:
study cannot be used for classification
Conclusions:
A valid conclusion cannot be drawn given the sketchy details of the methods and isolated nature of the results score.
Executive summary:

The study does not provide sufficient information on which to base a valid judgement of ocular irritancy potential.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 March 2012 to 26 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to current guidelines and GLP without significant deviation
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source:Harlan Italy s.r.l., San Pietro al Natisone, Italy
- Age at study initiation: 9-11 weeks old
- Weight at study initiation: 3.36 to 4.25 Kg
- Housing: Noryl cages with suspended perforated floors
- Diet (e.g. ad libitum): ad libitum Stanrab (P) SQC from Special Diet Services
- Water (e.g. ad libitum): ad libitum via cage bottles
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19+/-2
- Humidity (%): 55+/-15
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 12 March 2012 To: 15 March 2012
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg per eye
- Concentration (if solution): not applicable
Duration of treatment / exposure:
Single application on Day 1. The eyes were not rinsed to remove the test material from the eye after a fixed period of time.
Observation period (in vivo):
Rabbits were observed for 72 hours after instillation.
Number of animals or in vitro replicates:
Three. The sentinel was dosed first followed by the remaining two rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not done

SCORING SYSTEM: Draize system, according to test guideline

Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no corneal reactions observed
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: No iridial changes recorded
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: very slight erythema/conjnuctival redness recorded after one hour in one rabbit. Reactions had resolved by the 24 h assessment
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no reeaction observed
Irritant / corrosive response data:
No significant ocular irritation was observed in any of the three treated rabbits. Mean scores for all parameters measured over the 24-72 h post instillation period were 0.0. No classification is required for ocular irritancy, No signal word or Hazard Statement required in accordance with Regulation 1272/2008
Other effects:
None

No further information, no significant irritation effects observed in the study

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not an eye irritant
Executive summary:

Three rabbits were treated by instillation of 100 mg of undiluted 4,4-biphenol. The untreated eye acted as a concurrent control. Observations were recorded at 1, 24, 4 and 72 hours after instillation. Only one rabbit showed any changes with very slight conjunctival redness apparent at the first assessment point but resolved within 24 hours of dosing. No reactions were observed in the other two rabbits. The results of the in vivo test confirm the earlier in vitro assessment of severe irritation or corrosion, supporting the conclusion that no classification is required for 4,4 -biphenol and no signal word or Hazard statement is needed in accordance with the requirements of Regulation 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of the test item 4,4-Biphenol to irritate skin was investigated in an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model - EPISKIN. According to the histotoxic potential, when referred to the negative control, the test item is considered to have no effect on the test system (119.4% of mean cell viability when compared to negative control). The negative and positive controls gave the expected results and the study was accepted as valid. According to the criteria defined in the guidelines for this test (cell viability less than 50%), the test item is not considered to have irritant effect on the skin under the reported experimental conditions. Limited information on the lack of skin irritancy was obtained fom the absence of local irritation in a modern dermal toxicity study conducted in rats at the limit dose level.

 

An in vitro BCOP assay indicates the absence of corrosion or severe eye irritation; 4,4 -biphenol did not increase corneal opacity and no macroscopic evidence of opacity was recorded. Corneal permeability was also unaffected by treatment. Only minimal transient reactions were seen in a study of eye irritation in vivo. Older range-finding data are not considered to be sufficiently reliable in the absence of detailed methodology or results and only provide information relating to corneal effects.


Justification for selection of skin irritation / corrosion endpoint:
The in vivo study requirement was waived on the basis of no indication of skin irritation in the in vitro assay, confirmed by limited data from an older range-finding study and the results of the dermal toxicity investigation.

Justification for selection of eye irritation endpoint:
The in vivo results were negative for ocular irritation confirming the in vitro results obtained for corneal changes

Justification for classification or non-classification

Results from in vitro screening assays for skin and eye irritation indicate no potential for eliciting irritant responses and consequently classification is not warranted for biphenyl-4,4'-diol.