Caesium fluoride

Endpoint summary

Description of key information

Based on an WoE evaluation of available data with the registered substance as well as with another fluoride salt, the substance is considered irritant to the skin and corrosive to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 February 2017 to 01 May 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

The mean OD570 of the negative control group was below the assay acceptance criteria lower limit of 0.6. However, as the result was only marginally below the lower limit and the test item gave an unequivocal negative result it was considered unnecessary to repeat the test. This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 216091B008
Purity: 99.9%
Physical state/Appearance: White powder
Expiry Date: 01 September 2018
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 Hour post exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end point can be used to either confirm a non-irritant result or will be used to override the non irritant result.

Vehicle:

not specified

Details on test system:

Supplier: SkinEthic Laboratories, Lyon, France
Date received: 25 April 2017
EpiSkinTM Tissues (0.38cm2) lot number:17-EKIN-017
Maintenance Medium lot number:17-MAIN3-016
Assay Medium lot number :17-ESSC-016

Amount/concentration applied:

The test item was used as supplied. 2 mL of maintenance medium, warmed to approximately 37 C, was pipetted into the second column of 3 wells of the 12 well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls, triplicate tissues treated with 10 µL of saline served as the additional negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 C, 5% CO2 in air for 42 hours.

Duration of treatment / exposure:

Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

refer to full study report

Value:

ca. 101.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues was 101.5% after the 15 Minute exposure period and 42 Hours post exposure incubation period.
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied with the exception that the mean OD570 of the negative control group was below the assay acceptance criteria lower limit of 0.6. However, as the result was only marginally below the lower limit and the test item gave an unequivocal negative result it was considered unnecessary to repeat the test.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of this study, the test item does not require classification.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.  The relative mean viability of the test item treated tissues was 101.5% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. The test item was classified as non-irritant based on the results of this study.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

One hundred ninety-nine male Sprague-Dawley rats (Charles River Laboratories) weighing approximately 250 g were used for all experiments (Skin histamine assays, Skin distribution of fluoride, Skin histamine receptor assays, measurement of skin adenyl cyclase activity). The animals were housed, two per cage, under constant temperature and humidity conditions with a 12-h light-dark cycle maintained. They were provided with Purina lab chow and fluoride-free drinking water ad libitum. Experimental treatment animals were randomly assigned to one of 12 experimental conditions. All rats had an area of the dorsal skin (approximately 6.0 cm2) exposed by shaving off all hair in that region with an electric hair clipper 24 h prior to the treatment conditions; one group (N = 12) of shaved-only rats (SO) received no further treatment. Other groups of rats had an X=shaped pattern scratched at the skin test site, using a 20-gauge needle. Each arm of the X measured 2.5 cm, and uniformity of this abrasion through the epidermis was assured by using a celluloid template. One such epidermally abraded (EA) group (N = 14) was given no further treatment.
For each of the shaved-only (SO) or epidermally abraded (EA) rats a patch of saturated filter fiber was applied to the skin test site, covered with gauze, and affixed with waterproof tape. The groups had applied: (a) distilled water (SO = 22; EA = 23); (b) 0.5% NaCl (SO = 12; EA = 12); (c) 1% NaCl (SO = t 2; EA = 29); (d) NaF, 0.5 % in distilled water (SO = 14; EA = 12), or (e) NaF, 1.0 % in distilled water (SO = 14; EA = 23).
The skin patch was kept in place for 24h by which time the fiber was dry; it was then removed and biopsies were performed under general anesthesia (pentobarbital, 50 mg/kg, i.p.).

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

See above in materials and methods

Type of coverage:

other: patch of saturated filter fiber covered with gauze, and affixed with waterproof tape.

Preparation of test site:

shaved

Remarks:

and abraded

Vehicle:

water

Amount / concentration applied:

(a) distilled water (SO = 22; EA = 23);
(b) 0.5% NaCl (SO = 12; EA = 12);
(c) 1% NaCl (SO = t 2; EA = 29);
(d) NaF, 0.5 % in distilled water (SO = 14; EA = 12),
(e) NaF, 1.0 % in distilled water (SO = 14; EA = 23).

Duration of treatment / exposure:

24h

Observation period:

7 days

Irritation parameter:

edema score

Basis:

mean

Time point:

24 h

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

24 h

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

The only treatment condition that produced an overt change at the skin test site after 24 h was the application of 1% NaF to the abraded epidermis. In this case the abraded area had developed a linear distribution of small vesicles along both scratch lines. These vesicles persisted for approximately 7 days.
The 24h application of 0.5%NaF did not produce overt vesicle formation along the abrasion line of the epidermally abraded rat skin.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The direct application of sodium fluoride (0.5 and 1.0% in distilled water) to the abraded skin of rats produced effects ranging from superficial necrosis to oedema and inflammation (Essman et al., 1981).

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study need not be conducted because the substance is classified as irritating to eyes with risk of serious damage to eyes

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Handbook data - No guideline reported

Principles of method if other than guideline:

Handbook data - No details on the guideline reported

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Vehicle:

other: aqueous solution

Amount / concentration applied:

2% of NaF in aqueous solution

Remarks on result:

other: 2% aqueous solution to the eye caused corneal epithelial defects, necrotic areas in the conjunctiva; Injection subconjunctivally or into the anterior chamber caused corneal edema

Irritant / corrosive response data:

Application of a 2% aqueous solution to the eye caused corneal epithelial defects and necrotic areas in the conjunctiva. Injection subconjunctivally or into the anterior chamber caused corneal edema and a severe inflammatory reaction in the eye with hemorrhages in the iris.

Conclusions:

Sodium fluoride has been tested on rabbit eyes in several different ways. Application of a 2% aqueous solution to the eye caused corneal epithelial defects and necrotic areas in the conjunctiva. Injection subconjunctivally or into the anterior chamber caused corneal edema and a severe inflammatory reaction in the eye with
hemorrhages in the iris. Note this reference is also quoted in the WHO EHC criteria (FLUORIDES (EHC 227, 2002)).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

To meet skin irritation endpoint for cesium fluoride, an in vitro test with the test item cesium fluoride was launched following the reconstructed human epidermis model (OECD guideline 439).

The test item was concluded not irritant after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

However, sodium fluoride studies have shown some adverse effects to skin. For instance, the team of Essman et al. (1981) has demonstrated that direct application of sodium fluoride (0.5 and 1.0% in distilled water) to the abraded skin of rats produced effects ranging from superficial necrosis to oedema and inflammation.  

Due to this information, cesium fluoride has been classified as irritant chemical cat.2.

 

Eye irritation

No in vitro/in vivo test is currently available for cesium fluoride. Therefore, data from the source substance NaF was used.

A publication using 2 % NaF shows that F- is eye damaging. Grant et al., 1986 & 1993 applied NaF in aqueous solution to the eye of rabbits. The solution caused corneal epithelial defects and necrotic areas in the conjunctiva. Injection subconjunctivally or into the anterior chamber caused corneal edema and a severe inflammatory reaction in the eye with hemorrhages in the iris.

Hence, as a conservative approach cesium fluoride is considered to be eye damaging and no further studies are needed.

Justification for classification or non-classification

The test item was classified as irritant to the skin cat 2 and as corrosive to eyes cat 1 according to criteria of EU CLP.