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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 October 2009 to 26 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The test item cC6O4 ammonium salt is an anionic surfactant classified according to CLP Regulation (REGULATION (EC) No 1272/2008) as Skin Irritant 2.
The LLNA test indicates C6O4 ammonium salt as skin sensitizer.
However, as expressed in the OECD 429 guideline the LLNA may not be suitable to test irritants.
In scientific literature, the fact that irritancy augments the induction response and/or can sometimes lead to non-specific proliferation responses in the LLNA is known (Basketter et al., 2009;Montelius et al., 1998; Woolhiser et al., 1998; Basketter et al., 2007a,b,c).
This raises the possibility of false positive results in the original LLNA caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation) (Vohr and Ahr, 2005).
The possibility of false positive findings for irritant substances, including some type of surfactants, is reported in the OECD guideline 429.
Mehling et al (2007)reports that for the category of surfactants, which is generally regarded to be without a sensitization, unexpected positive results were observed in LLNA tests.The OECD 429 guideline indicates as limitation the use of LLNA for this category of chemicals.
Basing on the reasonings reported above and according to the OECD 429 guideline, which states it should be recognised that for classes substances that are potential confounders the use of OECD 406 may necessitate, it was deemed necessary to better assess the sensitization potential of the test item carrying out a further study.
A Buehler test (OECD 406) was performed in order to get reliable information on skin sensitizer properties of the test substance.

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:

ammonium 2,2-difluoro-2-{[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:

ammonium 2,2-difluoro-2-{[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): C6O4 cyclic
- Substance type: pure substance (dry salt)
- Physical state: white powder
- Analytical purity: 98.2%
- Impurities (identity and concentrations): NH4F= 0.02% (w/w), NH4Cl=0.25% (w/w), H2O=0.2% (w/w), Organic impurities= 1.3% (w/w)
- Composition of test material, percentage of components: not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 156/17
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: not reported
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy. Breeder: Charles River Germany
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 271 to 325 grams
- Housing: Stainless steel cages measuring 48x63x41 (during study) or 87x71x24 cm (during acclimatisation), with grid floor. Daily inspected and changed as necessary (at least 3 times/week)
- Diet (e.g. ad libitum): 8GP17 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy) ad libitum
- Water (e.g. ad libitum): drinking water supplied to each cage via a water bottle (ad libitum)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): Approximately 15 to 25 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

IN-LIFE DATES:
From: no data
To: the end of the experimental procedure.

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

MAIN Study - Induction:
50% test substance in sterile water

Day(s)/duration:

day 1-3 , day 8-10 and day 15-17

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

MAIN Study - Challenge:
50% test substance in sterile water

Day(s)/duration:

On day 29

No. of animals per dose:

PRELIMINARY SCREEN:
5 animals. Each animal dosed with 2 concentration of the test item.

MAIN STUDY:
test group: 20 animal
control group: 10 animals

Details on study design:

The study was divided into 2 distinct phases. The first of these consisted of a preliminary screen which was used to determine suitable test item concentrations to be used in the second phase. This second phase constituted the main study: the determination of the sensitisation potential of the test item.

Allocation to groups:
Animals were allocated to treatment groups prior to each phase of the study.

Preliminary screen:
Five animals were selected from those available and the flanks clipped free of hair. Each animal was dosed with 2 concentrations of the test item, 1 on either flank. A gauze patch measuring at least 20x20 mm was soaked with 0.4 ml of the selected concentration of the test item. This was then placed onto the selected treatment site. A strip of synthetic film was placed over the treated sites and the whole assembly was secured in position by encircling the trunk of the animal with a length of adhesive strapping.
All animals were treated in this manner such that a total of 5 concentrations (50, 20, 10, 5 and 1% in the selected vehicle, sterile water) of the test item were dosed each in duplicate. The adhesive strapping and patches were removed after approximately 6 hours contact with the skin. The treated sites were washed with lukewarm water to remove any remaining test item.
Approximately 24 and 48 hours after removal of the dressings, the treated sites were examined for signs of reaction to treatment. Each site was assessed and scored using the following scale:
No visible change : 0
Discrete or patchy erythema: 1
Moderate and confluent erythema: 2
Intense erythema and swelling: 3

Main study - Induction:
Animals were allocated to treatment to give a test group of 20 animals and a control group of 10 animals.
On the day of dosing (Day 1) the hair was clipped from the anterior region of the left flank of each animal. Animals of the test group were treated with the test item at a concentration of 50%. A gauze patch measuring 20x20 mm was soaked with 0.4 ml of the test item and placed onto the selected skin site. A strip of synthetic film was placed over the treated sites and the whole assembly was secured in position by encircling the trunk of the animal with a length of adhesive strapping. All animals of the test group were treated with the test item in this manner and animals of the control group were similarly treated with the selected vehicle (sterile water).
After an exposure period of 6 hours the dressings were removed. The treated sites were cleaned of remaining test item or vehicle by washing with lukewarm water.
Approximately 24 and 48 hours after removal of the patches the treated sites were examined for signs of reaction to treatment. Each site was assessed and scored using the following scale:
No visible change : 0
Discrete or patchy erythema: 1
Moderate and confluent erythema: 2
Intense erythema and swelling: 3
These procedures were repeated at weekly intervals (Days 8 to 10 and 15 to 17 of the study).

Main study - Challenge
On Day 29, the hair was removed with electric clippers from both the anterior and posterior regions of the right flank of all animals of both test and control groups.
A 0.4 ml aliquot of the test item at a concentration of 50% was spread evenly over an absorbent patch measuring approximately 20x20 mm. This was placed onto the skin of the posterior region of the prepared site on the right flank. A similar patch, containing 0.4 ml of the vehicle selected for the challenge (sterile water), was placed onto the anterior region of the prepared site. A strip of synthetic film was placed over the treated sites and the whole assembly was secured in position by encircling the trunk of the animal with a length of adhesive strapping. All animals of the test and control groups were treated with both the test item and vehicle in this manner. After an exposure period of approximately 6 hours the dressings were removed and the treated sites cleaned of the remaining test item by washing with lukewarm water.
Approximately 21 hours after removal of the dressing and patches, the treated sites were closely clipped to remove any hair that may have grown. Approximately 3 hours later, 24 hours after removal of the dressing, the treated sites were examined for any signs of reaction to treatment.
The degree of skin reaction was scored according to the following scheme:
No visible change : 0
Discrete or patchy erythema: 1
Moderate and confluent erythema: 2
Intense erythema and swelling: 3
Skin reaction on the treated sites was again assessed approximately 24 hours after the first examination (approximately 48 hours after removal of the patches).

Termination and necropsy:
All animals were killed by carbon dioxide narcosis following the end of the experimental procedure. No necropsy examination was performed on these animals.

Challenge controls:

The same concentration of test item (50% in sterile water) was selected for the challenge as no irritation was noted in all main phase animals.
No response was observed to the test item at the selected concentration, in either test or control group animals 24 and 48 hours following 6 hours topical exposure. No reaction was observed to the vehicle alone.

Positive control substance(s):

yes

Remarks:

A positive control check using alpha-Hexylcinnamaldehyde is performed approximately at 6 monthly intervals.

Results and discussion

Positive control results:

A positive control check using alpha-Hexylcinnamaldehyde is performed approximately at 6 monthly intervals.
A summary relevant to the most recent reliability check (RTC STUDY NUMBER: 28130-007INT) performed follows:

REFERENCE SUBSTANCE: α-HEXYLCINNAMALDEHYDE
CONCENTRATION INDUCTION: 70% in DMSO
CONCENTRATION CHALLENGE: 15% in acetone
CRITICAL DATES:
INDUCTION: 23 March 2009, 30 March 2009, 06 April 2009
CHALLENGE: 20 April 2009

RESULTS: 20 % response in test group and 0 % response in control group at challenge
INTERPRETATION: Incidence at challenge acceptable
Test system regarded as valid.

In accordance with OECD TG 406 the reliability check of the model to confirm sensitivity and reliability of the technique was performed every 6 month with a positive control substance (Alpha-Hexylcinnamaldehyde). The number of animals tested with the positive control were not reported, but were assumed to follow recommendations for positive control (minimum 10 animals for control groups).

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.4 ml of sterile water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no reported effects

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.4 ml of sterile water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no reported effects

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4 ml of a 50% aqueous solution

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no reported effects

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.4 ml of a 50% aqueous solution

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no reported effects

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

15%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

no reported effects

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

15 %

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

no reported effects

Any other information on results incl. tables

This table details the findings at the treated sites on the left flank of each animal 24 and 48 hours following 6 hours topical exposure to the test item, C6O4 cyclic, at a concentration of 50% and the vehicle alone (sterile water) during the induction procedure:

Group function	Animal number	Dermal Response
		First Induction		Second Induction		Third Induction
		24 hours	48 hours	24 hours	48 hours	24 hours	48 hours
CONTROL	181	0	0	0	0	0	0
	183	0	0	0	0	0	0
	185	0	0	0	0	0	0
	187	0	0	0	0	0	0
	189	0	0	0	0	0	0
	191	0	0	0	0	0	0
	193	0	0	0	0	0	0
	195	0	0	0	0	0	0
	197	0	0	0	0	0	0
	199	0	0	0	0	0	0
TEST	201	0	0	0	0	0	0
	203	0	0	0	0	0	0
	205	0	0	0	0	0	0
	207	0	0	0	0	0	0
	209	0	0	0	0	0	0
	211	0	0	0	0	0	0
	213	0	0	0	0	0	0
	215	0	0	0	0	0	0
	217	0	0	0	0	0	0
	219	0	0	0	0	0	0
	221	0	0	0	0	0	0
	223	0	0	0	0	0	0
	225	0	0	0	0	0	0
	227	0	0	0	0	0	0
	229	0	0	0	0	0	0
	231	0	0	0	0	0	0
	233	0	0	0	0	0	0
	235	0	0	0	0	0	0
	237	0	0	0	0	0	0
	239	0	0	0	0	0	0

KEY: 0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

This table details the findings at the treated sites on the right flank of each animal 24 and 48 hours following challenge by 6 hours topical exposure to the test item, C6O4 cyclic, at a concentration of 50% and the vehicle alone (sterile water):

Group function	Animal number	Dermal Response
		Vehicle		Test item
		24 hours	48 hours	24 hours	48 hours
CONTROL	181	0	0	0	0
	183	0	0	0	0
	185	0	0	0	0
	187	0	0	0	0
	189	0	0	0	0
	191	0	0	0	0
	193	0	0	0	0
	195	0	0	0	0
	197	0	0	0	0
	199	0	0	0	0
TEST	201	0	0	0	0
	203	0	0	0	0
	205	0	0	0	0
	207	0	0	0	0
	209	0	0	0	0
	211	0	0	0	0
	213	0	0	0	0
	215	0	0	0	0
	217	0	0	0	0
	219	0	0	0	0
	221	0	0	0	0
	223	0	0	0	0
	225	0	0	0	0
	227	0	0	0	0
	229	0	0	0	0
	231	0	0	0	0
	233	0	0	0	0
	235	0	0	0	0
	237	0	0	0	0
	239	0	0	0	0

KEY: 0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No response to the test item, at the selected concentration, was apparent at challenge in any animal of the test and control groups.
These results indicate that the test item cC6O4 ammonium salt does not elicit a sensitisation response in the guinea pig, being there no evidence of response at challenge following a period of induction exposure to the test item.

Executive summary:

The potential of the test item, cC6O4 ammonium salt, to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the methods of Buehler.

The concentrations of the test item used in the main study were determined by the results of a preliminary screening test. The main sensitisation test was undertaken using a test group of 20 animals and a control group of 10 animals. In an attempt to induce sensitisation, test animals were treated by topical application of the test item at a concentration of 50% in sterile water. This was repeated at weekly intervals for a total of 3 weeks. Animals of the control group were treated in the same manner but the vehicle alone (sterile water) was used in place of the test item. Two weeks after the third and final induction exposure, animals of the test and control groups were challenged by topical application of both the test item and the vehicle alone (sterile water).

At challenge no response was observed for the test item at a concentration of 50% in either test or control group animals. No reaction was observed to the vehicle alone (sterile water).

These results indicate that the test item cC6O4 ammonium salt does not elicit a sensitisation response in the guinea pig, being there no evidence of response at challenge following a period of induction exposure to the test item.