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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-Jan-2010 to 02-Feb-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD and EU guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,3,3-tetrafluorooxetane
EC Number:
700-202-5
Cas Number:
765-63-9
Molecular formula:
C3H2F4O
IUPAC Name:
2,2,3,3-tetrafluorooxetane
Details on test material:
- Name of test material (as cited in study report): 2,2,3,3-tetrafluorooxetane (TFO)
- Physical state: Liquid
- Analytical purity: 99%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: Not reported
- Lot/batch No.: TFOS285001
- Expiration date of the lot/batch: 30 April 2010
- Stability under test conditions: Not stated
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: a reputable supplier of laboratory animals
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 18.4 to 23.2 g
- Housing: barriered rodent facility, individually in polycarbonate cages with woodflake bedding
- Diet (e.g. ad libitum): standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) ad libitum
- Water (e.g. ad libitum): Potable water taken from the public supply ad libitum
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 19 January 2010 To: 01 February 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 & 50% v/v TFO in vehicle
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Irritation: The maximum practical concentration for pinna dosing was 50% v/v in (Acetone:olive oil 4:1 v/v). Based on this information the following concentration was selected for the preliminary investigation: 25 and 50% v/v
The results of the preliminary investigation indicated that 50% v/v was a suitable high dose level for the main phase of the study. Based on this information the following concentrations were selected for the main phase of the study: 10, 25 and 50% v/v
- Lymph node proliferation response: Not determined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled treatment group approach
- Criteria used to consider a positive response:
The test substance is regarded as a sensitizer if at least one concentration of the test substance
results in a three-fold greater increase in 3HTdR incorporation compared to control values

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 μl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette.
Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered
saline containing 3H-methyl Thymidine (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.
Five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group.
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting.
The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A study was performed to confirm the sensitivity and reliability of the experimental technique used at Huntingdon Life Sciences to detect skin sensitization potential. The study was performed using the murine local lymph node assay (OECD 429 individual animal approach) and a known moderate sensitizer – hexyl cinnamic aldehyde (HCA).
The positive control study is considered to be valid if the results from the HCA group have a three-fold greater increase in 3HTdR incorporation compared to control values.
In this assay the test/control ratio obtained for HCA at 25% (HCA: vehicle, v/v, vehicle as for the main test). This indicates that HCA demonstrates the potential to induce skin sensitization (delayed contact hypersensitivity) and confirms the sensitivity of the technique to detect sensitization potential.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Not reported
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group 5069.60 10% TFO 11733.90 25% TFO 14060.50 50% TFO 10066.60

Any other information on results incl. tables

Table 1 gives the results for dpm/node and test/control ratios obtained with TFO.

Table 1 Group dpm/node and test/control ratios

Group

Concentration % v/v

dpm

Number of lymph nodes per group

dpm/node

Test/control ratio†

Result

+ = positive

- = negative

3

0

5069.60

8.0

633.70

n/a

n/a

4

10

11733.90

7.0

1676.27

2.6

-

5

25

14060.50

8.0

1757.56

2.8

-

6

50

10066.60

8.0

1258.33

2.0

-

† Test/control of 3 or greater indicates a positive result

n/a Not applicable         

dpm Disintegrations per minute (less background count of 44.70 dpm)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
TFO is not regarded as a potential skin sensitizer.

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