Reaction mass of potassium difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate with potassiumfluoride and potassium carbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

31 March 2009 to 30 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is guideline conforme (OECD 429) under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2009-03-30, Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

cC6O4 ammonium salt

IUPAC Name:

cC6O4 ammonium salt

Constituent 2

Reference substance name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

IUPAC Name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Constituent 3

Reference substance name:

Reaction mass of ammonium difluoro {[(4S,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4R,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4S,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate and ammonium difluoro {[(4R,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

EC Number:

682-238-0

Cas Number:

1190931-27-1

Molecular formula:

C6H4F9NO6

IUPAC Name:

Reaction mass of ammonium difluoro {[(4S,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4R,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate, ammonium difluoro {[(4S,5S)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate and ammonium difluoro {[(4R,5R)-2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

Details on test material:

- Name of test material (as cited in study report): cC6O4
- Molecular formula (if other than submission substance): C6H4F9NO6
- Molecular weight (if other than submission substance): 357
- Smiles notation (if other than submission substance): [NH4+].FC1(F)OC(F)(OC(F)(F)C([O-])=O)C(F)(OC(F)(F)F)O1
- InChl (if other than submission substance): no data
- Structural formula attached as image file (if other than submission substance): see Fig.1 (C6O4 Structural formula)
- Substance type: pure substance
- Physical state: solid
- Analytical purity: 96.2% as ammonium salt
- Impurities (identity and concentrations): NH4F=0.021% (w/w), NH4Cl=0.31% (w/w), H2O=0.328% (w/w), the organic impurities are dioxolan based and represent the 0.8% by mol on total mol of organic phase of sample
- Composition of test material, percentage of components: not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 150/31
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: not reported
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: mean 20.2 ± 0.9 g; range 18.5 - 22.0 g
- Housing: group housing; cage Makrolon Type II, with wire mesh top; granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum,
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature 22 + 2°C
- Humidity (%): relative humidity 45-65%
- Air changes (per hr): The experiment was conducted under standard laboratory conditions.
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.


IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

vehicle for positive control: acetone:olive oil (4+1)

Concentration:

test item concentrations of 10, 25 and 50% (w/v) in propylene glycol

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 50 % solution in dimethylformamide. THowever, propylene glycol was used as recommended by the sponsor. the same solubility was observed.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated with concentrations of 25 and 50% each on three consecutive days. In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 10, 25 and 50%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
- Lymph node proliferation response: not reported


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared balance and propylene glycol was quantitatively added. The preparations were made freshly before each dosing occasion.
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25 and 50% (w/v) in propylene glycol. The application volume, 25 µl, was spread over the entire dorsal surface (  8 mm) of each ear lobe once daily for three consecutive days. Three further group of mice were treated with an equivalent volume of the test item vehicle (propylene glycol), vehicle for the positive control item (acetone:olive oil (4+1)) or the positive control item (25% -Hexylcinnamaldehyde).
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 µl of 80.8 µCi/ml 3HTdR (corresponds to 20.2 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, D-30827 Garbsen).
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

DPM per lymph node: 3689.3
SI: 10.22

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle positive control: 1.0 Vehicle test item: 1.0 10% test item: 2.32 25 % test item: 2.05 50% test item: 4.72 Positive control: 10.22

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: Vehicle positive control: 361.2 Vehicle test item: 217.4 10% test item: 503.6 25 % test item: 446.5 50% test item: 1025.4 Positive control: 3689.3

Any other information on results incl. tables

EC3 computation: EC3 = (a-c) [(3-d)/(b-d)] + c =33.9%(w/v)

a = 25, b = 2.05, c = 50, d = 4.72

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item cC6O4 ammonium salt was found to be a weak skin sensitiser under the described conditions.
Considering the similarity of chemical structure between F- Diox potassium salt and cC6O4 ammonium salt, it can be concluded that a similar bahaviour for F- Diox potassium salt is expected.

Executive summary:

In order to evaluate the skin sensitization properties of F- Diox potassium salt it was deemed appropriate to use the Read Across approach based on the experimental studies performed on cC6O4 ammonium salt by virtue of similarity of chemical structure between F- Diox potassium salt and cC6O4 ammonium salt (CAS no: 1190931-27-1).

F-Diox potassium salt and cC6O4 ammonium salt are two salts of the same carboxylic acid, they differ only for the cationic part (K+ in F-Diox potassium salt and NH4+ in cC6O4 ammonium salt). Considering the similarity of chemical structure a similar biological behaviour is expected.

According to column 2 of section 8.3 of Annex VII the LLNA was performed as a first choice on the test item.

The purpose of this study was to evaluate the skin sensitizing potential of test item cC604 ammonium salt. The study followed the protocol according to OECD Guideline 429.

In the study the test item cC6O4 ammonium salt dissolved in propylene glycol was assessed for its possible contact allergenic potential.

The LLNA was performed using test item concentrations of 10, 25 and 50%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 2.32, 2.05 and 4.72 were determined with the test item at concentrations of 10, 25 and 50% in acetone:olive oil (4+1), respectively.

The results of this study indicate C6O4 ammonium salt as skin sensitizer under test conditions. Following the criteria reported in the technical report ECETOC 87 (2003), which proposes criteria to classify skin sensitizer according to potency, the result of this test indicates C6O4 as weak sensitizer since an EC3 value of 33.9% was calculated.