Cesium fluoroaluminate

Endpoint summary

Description of key information

Skin sensitisation: not sensitising (modified OECD 429; GLP)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

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 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010-07-22

Deviations:

yes

Remarks:

modified OECD 429, method according to Ehlings et al. 2005

Principles of method if other than guideline:

The test was performed in accordance with the method according to Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count (i.e. sensitising properties) and ear weight (i.e. irritating properties) were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (lymph node cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, store in a tightly closed container in a dry and well-ventilated place

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 - 12 weeks
- Weight at study initiation: weight variation < 20 % of mean
- Housing: kept singly in MAKROLON cages (type II) with a basal surface of approx. 360 cm² and a height of approx. 14 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany); animals are not group-housed to prevent contact of the application sites.
- Diet (ad libitum): ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 10 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

10 %, 25 % and 50 % (w/w) of the test item

No. of animals per dose:

6 females

Details on study design:

RANGE FINDING TEST
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc.
A 50% suspension was the highest feasible concentration of cesium fluoroaluminate in propylene.

MAIN STUDY
The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal was individually identified. The weights and any clinical observation were recorded. In addition, ear swelling measurements were carried out
at the helical edge of both ears using an Oditest micrometer. Open application of 25 μL of the appropriate dilution of the test substances, the vehicle alone, or the positive control (as appropriate), to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4 (24 h after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. Lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS:
- Clinical signs: animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. In addition, animals were checked regularly throughout the working day and on the weekend.
- Body weight: the weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS
The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p ≤ 0.01 is considered positive, however, an increase in lymph node weight is an indication for possible irritating properties not sensitising properties.
In addition, the average ear weights per group and the average ear thickness per group were compared to the vehicle control group as an indication for possible irritaitng properties. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Please refer to "details on study design"

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.
positive control: SI: 1.600 (lymph node weight); SI: 1.185 (ear thickness)
positive control (vehicle): SI: 1.000 (lymph node weight); SI: 1.000 (ear thickness)

Key result

Parameter:

SI

Value:

1.595

Test group / Remarks:

10 % test item

Remarks on result:

other: lymph node cell count

Key result

Parameter:

SI

Value:

1.085

Test group / Remarks:

10 % test item

Remarks on result:

other: ear weight

Key result

Parameter:

SI

Value:

1.887

Test group / Remarks:

25 % test item

Remarks on result:

other: lymph node cell count

Key result

Parameter:

SI

Value:

1.177

Test group / Remarks:

25 % test item

Remarks on result:

other: ear weight

Key result

Parameter:

SI

Value:

2.69

Test group / Remarks:

50 % test item

Remarks on result:

other: lymph node cell count

Key result

Parameter:

SI

Value:

1.277

Test group / Remarks:

50 % test item

Remarks on result:

other: ear weight

Parameter:

SI

Value:

1

Test group / Remarks:

negative control group

Remarks on result:

other: lymph node cell count

Parameter:

SI

Value:

1

Test group / Remarks:

negative control group

Remarks on result:

other: ear weight

Parameter:

SI

Value:

1.488

Test group / Remarks:

positive control group

Remarks on result:

other: lymph node cell count

Parameter:

SI

Value:

1.213

Test group / Remarks:

positive control group

Remarks on result:

other: ear weight

Parameter:

SI

Value:

1

Test group / Remarks:

positive control group vehicle

Remarks on result:

other: lymph node cell count

Parameter:

SI

Value:

1

Test group / Remarks:

positive control group vehicle

Remarks on result:

other: 1.000

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method).

RESULTS ON SKIN SENSITISATION
In the main study treatment with cesium fluoroaluminate at concentrations of 10 %, 25 % or 50 % revealed increased values for the lymph node cell count (statistical significantly at 25 % and 50 % (p ≤ 0.01)). The stimulation index of the lymph node cell count of 1.4 was exceeded markedly at all tested concentrations.
In addition, increases were noted for lymph node weights for all concentrations. At the 25% and the 50% concentrations the stimulation indices of ear weight exceeded the threshold level of 1.1, the test item was considered to have irritating properties in this concentration range in this test system. However, as the 10% concentration did not exceed the threshold level of 1.1, the test item possesses sensitising potential at this concentration.


STIMULATION INDEX RESULTS OF LYMPH NODE WEIGHT AND EAR THICKNESS
10 % w/w: SI: 1.082 (lymph node weight); SI: 1.012 (ear thickness)
25 % w/w: SI: 1.163 (lymph node weight); SI: 1.091 (ear thickness)
50 % w/w: SI: 1.551 (lymph node weight); SI: 1.107 (ear thickness)
negative control: SI: 1.000 (lymph node weight); SI: 1.000 (ear thickness)

CLINICAL OBSERVATIONS:
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS
The animal body weight was not affected by the treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

Cesium tetrafluoroaluminate was tested with a modified LLNA test according to Ehling et al. (2005) in concentrations of 10, 25 and 50 % to determine the sensitising potential of the substance. During the assay the proliferation of the lymphocytes are measured using the parameters lymph node cell count and lymph node weight, whereas the former indicates a sensitizing property of the substance, if a stimulation index of 1.4 or greater is calculated. The latter parameter also provides information on sensitising properties, however, a statistical significant increase in lymph node weight is an indication of possible irritating not sensitizing properties.

In addition to the parameters, as stated above, ear weight and ear thickness are also recorded during testing. These parameters measure the irritating potential of a substance. If the ear weight results in a stimulation index of 1.1 or greater the substance is considered to be irritating to the skin and not sensitising.

Considering the results of the current study, all tested concentrations gave a stimulation index of above 1.4 for lymph node cell count, which obviously demonstrates skin sensitising potential of the substance. However, the stimulation index calculated for the ear weight was 1.1 or greater in all dose groups. Therefore, it can be concluded that the substance shows a clear irritating potential within the utilised test system which makes it impossible to distinguish between irritating and sensitising properties. Hence, a sensitising potential of the test substance cesium tetrafluoroaluminate cannot be identified free of doubt.

Based on the findings of the current study, cesium tetrafluoroaluminate is not considered to be a skin sensitiser due to the skin irritating potential that was observed during testing within the test system. The consideration is support by the knowledge that cesium, aluminium and fluoride are not classified as skin sensitiser and synergistic effects of the elements are not known and are not expected.

Hence, the local lymph node assay is considered to be not suitable for cesium tetrafluoroaluminate. Even OECD guideline 429 (2002) itself explicitly notes that the LLNA may not be suitable to test metals and irritants: „Despite the advantages of the LLNA over traditional guinea pig tests, it should be recognised that there are certain limitations that may necessitate the use of traditional guinea pigs tests (e.g., false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants)“. For example, in a comparison with the classification based on human experience, the LLNA did not correctly identify nickel (false negative) and copper (false positive) (Basketter et al. 1999).

In order to gain more information on a possible skin sensitising property of the substance, a Guinea pig maximization test according to the OECD 406 (1992) or an adequate alternate test system could be used to provide more conclusive information.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-01-15 to 2019-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992-07-17

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2017-05-08

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The purpose of this study was to determine the potential of Cesium fluoroaluminate to provoke skin sensitisation reactions in guinea pigs.
It should be noted that according to ECHA guidance on information requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance Draft Version 5.0 - June 2016: “The Murine Local Lymph Node Assay (LLNA), allowing assessment of potency, is the first-choice method for in vivo testing in case new in vivo testing is needed.” However, as stated it the OECD method 429: “…certain limitations that may necessitate the use of TG 406 (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants…”.
The test item cesium fluoroaluminate is a metal substance. Hence, the GPMT (OECD 406) was performed.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in the tightly closed original container and in a well-ventilated place

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 35 days
- Weight at study initiation: 304 - 360 g (excluding positive control group); positive control group: 320 - 359 g
- Housing: kept in groups of 5 in MAKROLON cages (MZK 80/25); bedding material: granulated textured wood (Granulat A2, J. BRANDENBURG, 49424 Goldenstedt, Germany)
- Diet (ad libitum): commercial diet, ssniff® Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 10 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Concentration / amount:

0.1 mL of Freund's complete adjuvant (diluted 1:1 with 0.9 % NaCl)

Day(s)/duration:

Day 0

Adequacy of induction:

other: since the test item (75 % concentration) was only slightly irritating, the exposed skin was coated with 10 % SDS on day 6

Route:

intradermal

Vehicle:

other: sesame oil

Concentration / amount:

0.1 mL of 0.01 % cesium fluoroaluminate

Day(s)/duration:

Day 0

Adequacy of induction:

other: since the test item (75 % concentration) was only slightly irritating, the exposed skin was coated with 10 % SDS on day 6

Route:

intradermal

Vehicle:

other: sesame oil

Concentration / amount:

0.1 mL of cesium fluoroaluminate in a 1:1 mixture (v/v) Freund's complete adjuvant/physiological saline

Day(s)/duration:

Day 0

Adequacy of induction:

other: since the test item (75 % concentration) was only slightly irritating, the exposed skin was coated with 10 % SDS on day 6

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

75 % cesium fluoroalumnate

Day(s)/duration:

Day 7 (exposure: 48 hours)

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

50 % cesium fluoroaluminate

Day(s)/duration:

Day 21 (exposure period: 24 hours)

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 (test substance) + 5 (controls)

Details on study design:

RANGE FINDING TESTS:
Eight male Dunkin-Hartley guinea pigs (age: 33 days) were used for the preliminary test: 6 animals for the topical administration and 2 animals for the intracutaneous administration.

The allocation of different test sites of the animals was alternated in order to minimize site-to-site variations in response.
The shoulder and the flank region of the animals were shaved (approx. 5 cm x 5 cm). Animals, even if only slightly injured, were replaced.
Sesame oil was used as vehicle.
(a) intracutaneous (72 hour observation period):
0.1 mL of the prepared test item was administered intracutaneously (shoulder region).
Three concentrations (1, 5 and 10 %) of the test item were injected intradermally into one, 3 further concentrations (0.01, 0.1 and 0.5 %) into a second animal.

(b) topical:
The test area of the animals each was shaved. 2 mL of the test preparation was spread over a filter paper (2 cm x 4 cm) and applied to the test area and held in contact by an occlusive dressing.
Two concentrations each were applied to the flanks of each animal (two animals: 50 and 75 %; two animals: 10 and 25 %; two animals: 1 and 5 %)
The occlusive dressing and the filter paper containing the test item were removed after 24 or 48 hours from respective 3 animals and the application sites were assessed immediately, 24 and 48 (24-hour exposure) or immediately and 24 hours (48-hour exposure) after removal of the filter paper for erythema and oedema using the Magnusson and Kligman grading scale.

Results:
(a) intracutaneous:
The concentration of 0.01% revealed a moderate and confluent erythema 24 and 48 hours, a discrete or patchy erythema 72 hours after administration. Concentrations of 0.1% and above revealed an intense erythema and swelling 24, 48 and 72 hours after administration.

(b) topical:
No skin reactions were observed at the concentration of 50%. The concentration of 75% revealed a discrete or patchy erythema 24 or 48 hours to 72 hours after start of exposure.

Hence, it was decided to use a 0.01% suspension for the 1st (intracutaneous) induction stage, a 75% suspension for the 2nd (topical) induction stage and a 50% suspension for the challenge in the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and dermal application)
- Site: shoulder region
- Frequency of applications: three pairs of intradermal injections were given in the shoulder region once and 6 days later the skin was treated with sodium lauryl sulfate 10 % in vaseline in order to induce a local irritatrion. 24 hours later the test item was topically applied using a patch-test technique.
- Concentrations:
Test animals:
Intradermal:
i) 0.1 mL FCA diluted 1:1 with 0.9 % NaCl
ii) 0.1 mL test item in sesame oil (final concentration: 0.01 % (w/w))
iii) 0.1 mL test item in FCA mixed with physiological saline in the ratio 1:1 (final concentration: 0.01 % (w/w))
Topical application:
75 % concetration of test item

Control animals:
Intradermal injection:
i) 0.1 mL FCA diluted 1:1 with aqua ad iniectabilia
ii) 0.1 mL aqua ad iniectabilia
iii) 0.1 mL aqua ad iniectabilia and FCA/physiological saline (1:1) in the ratio 1:1 (w/w)
Topical application:
sesame oil

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (two weeks after the topical application (induction))
- Exposure period: 24 hours
- Site (test animals and controls): both flanks shaved; right flank: sesame oil; left flank: 50 % of the test item in sesame oil
- Evaluation (hr after challenge): 24 and 48 hours

OTHER OBSERVATIONS:
- mortality: daily during the observation period
- clinical signs: daily during the observation period
- body weight: at start of study and at study termination

Challenge controls:

5 guinea pigs were used as control animals.
Challenge dose: right flank: sesame oil; left flank: 50 % of the test item in sesame oil

Positive control substance(s):

yes

Remarks:

α-hexyl cinnamic aldehyde (historical background group from October/November 2018; 20 animals; intradermal (induction): 10 %; topical (induction): undiluted; challenge: 5 %)

Positive control results:

Animals treated with α-hexyl cinnamic aldehyde in sesame oil exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema (grade 1) 48 and 72 hours after start of exposure.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % suspension of test item in sesame oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % suspension of test item in sesame oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % suspension of test item in sesame oil

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % suspension of test item in sesame oil

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5 % of α-hexyl cinnamic aldehyde

No. with + reactions:

20

Total no. in group:

20

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5 % of α-hexyl cinnamic aldehyde

No. with + reactions:

20

Total no. in group:

20

MAIN STUDY -INDUCTION

A 0.01% suspension of cesium fluoroaluminate in sesame oil chosen for the 1st (intracutaneous) induction stage revealed a moderate and confluent erythema 24 and 48 hours after administration in all 10 test item-treated animals.

Two mL of the 75% concentration of cesium fluoroaluminate/animal chosen for the second (topical) induction stage were only slightly irritating to the shaved skin in the preliminary test. Hence, the skin was coated with sodium lauryl sulfate on the day before stage 2 induction in order to induce a local irritation. This treatment resulted in a moderate and confluent erythema 48 and 72 hours after start of exposure in all animals.

OBSERVATIONS:

The body weight gain of the animals treated with Cesium fluoroaluminate was within the range of the vehicle control during the experiment.

Behaviour remained unchanged during the course of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the Regulation (EC) 1272/2008 and subsequent adaptations, the substance is not sensitising to the skin.
Furthermore, test results indicate no skin irritation potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Skin sensitisation

The substance does not possess a skin sensitisation potential and does not require classification as skin sensitiser according to Regulation (EC) No 1272/2008 and subsequent adaptations.