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

Endpoint summary

Description of key information

Two different experimental studies are reported: a Local lymph node assay (LLNA) and a Buehler test.
The onset of false positive results for irritant substances in the LLNA is discussed, since the test item C6O4 ammonium salt is an anionic surfactant classified as Skin Irritant 2 according to CLP Regulation (REGULATION (EC) No 1272/2008).

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:

supporting study

Study period:

31 March 2009 to 30 October 2009

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)

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)

Remarks:

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

Type of study:

mouse local lymph node assay (LLNA)

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:

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. However, 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 (d = 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% alfa-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.

Positive control results:

DPM per lymph node: 3689.3
SI: 10.22

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle of positive control

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle of test item

Key result

Parameter:

SI

Value:

2.32

Test group / Remarks:

10% test item

Key result

Parameter:

SI

Value:

2.05

Test group / Remarks:

25% test item

Key result

Parameter:

SI

Value:

4.72

Test group / Remarks:

50% test item

Key result

Parameter:

SI

Value:

10.22

Test group / Remarks:

Positive control

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

361.2

Test group / Remarks:

Vehicle of positive control

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

217.4

Test group / Remarks:

Vehicle of test item

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

503.6

Test group / Remarks:

10% test item

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

446.5

Test group / Remarks:

25% test item

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

1 025.4

Test group / Remarks:

50% test item

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

3 689.3

Test group / Remarks:

Positive control

Key result

Parameter:

EC3

Value:

33.9

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.

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 skin sensitiser under the described conditions.

Executive summary:

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 cC6O4 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 cC6O4 ammonium salt as weak sensitizer since an EC3 value of 33.9% was calculated.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

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.

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.

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

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.

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).

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

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

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Two in vivo studies for skin sensitisation are reported: a Murine Local Lymph Node Assay (LLNA) (OECD 429) and Guinea Pig test (OECD 406)

In order to evaluate the potential for skin sensitization of cC6O4 ammonium salt, according to column 2 of section 8.3 of Annex VII of REACH Regulation, the LLNA was performed as a first choice on the test item.

 

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 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 ammonium salt as weak sensitizer since an EC3 value of 33.9% was calculated.

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) reported 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.

 

The Buehler (OECD 406) test was performed since the method well reproduces the normal human exposure through dermal contact.

In the Buehler test a net response of 15% incidence is used as the criterion for a chemical being classified as a sensitizer.

C6O4 tested in the Buehler test at the higher non irritant concentration (concentration of 50% in sterile water) both for induction and challenge phases, showed 100% negative results: no response to the test item was apparent at challenge in any animal of the test and control groups. This result indicates the test item is a non sensitizer

 

In the light of these results and considering the limitation of LLNA with irritant substances, the LLNA result for C6O4 ammonium salt can be interpreted as a false positive.

Therefore it can be affirmed that cC6O4 ammonium salt does not have skin sensitising properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the results and the considerations reported above, it is concluded that the cC6O4 ammonium salt is not a skin sensitiser. The REGULATION (EC) No 1272/2008 (EU Regulation on Classification, Labelling and Packaging of substances and mixtures) would indicate the following for cC6O4 ammonium salt:

 

SKIN SENSITISATION

Classification: not required

Signal word: none indicated

Hazard statement: none indicated