(E)-4-Ethoxy-1,1,1-trifluoro-3-buten-2-one (stabilized with BHT)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Feb.-05 Mar. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately characterised with its purity. Therefore full validation applies.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Remarks:

The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Details on test material:

N.B. This batch BWG 60000 was used in several studies with some descriptive information diverging slightly between the reports (for further details, see below in this field and in the following field).

- Appearance:
* Clear yellowish liquid was mentioned in the 2007 and 2008 Notox reports dealing with physico-chemical properties and in the in 2007 Solvay Pharma report dealing with ASRI test.
* Yellow liquid was mentioned in the 2006 and 2007 TNO reports dealing with toxicological endpoints.
- Storage conditions:
* Storage in freezer (=< -15°C) in the dark under nitrogen was mentioned in the 2007 and 2008 Notox reports dealing with physico-chemical properties.
* Storage in freezer (< -10°C) was mentioned in the 2007 Solvay Pharma report dealing with ASRI test.
* Storage at < -18°C (with or without the mention "protected from light") was mentioned in the 2006 and 2007 TNO reports dealing with toxicological endpoints.
- Stability under storage conditions: Stable.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan, The Netherlands.
- Females nulliparous and non-pregnant: yes.
- Microbiological status of animals: During the quarantine period serological investigation of the microbiological status was conducted in a few, randomly selected animals. The results of serological examinations indicated an acceptable microbiological status and the animals were accepted for use in the study.
- Age at arrival: approximately 6 weeks.
- Weight at study initiation: 19.94 g (mean weight of the 25 mice used for the main study).
- Housing: group housing in macrolon cages (Type VI, maximally 5/cage), from the start of treatment. After allocation the animals were housed individually (macrolon Type II cages).
- Diet: Feed was provided ad libitum from the arrival of the mice until the end of the study. The mice were fed a commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3). Each batch used at TNO has been analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants. The certificate of analysis pertaining to the batch used in this study (batch 5385) was retained in the archives of TNO Quality of Life. The results of the analysis were within acceptable limits. The feed was provided as pellets.
- Water: Drinking water was provided ad libitum from the arrival of the mice until the end of the study. The drinking water (tap water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens. The supplier routinely examined the physical, chemical and microbiological variables of the drinking water. In addition, the supplier periodically (twice per year) analysed water samples taken at the premises of TNO Quality of Life in Zeist for a limited number of physical, chemical and microbiological variables. The results of the routine examinations and of the samples taken were made available to and archived at TNO Quality of Life.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C.
- Humidity: 40-70%, a single deviation from the optimal range was observed (minimal value 39.9% for less than one hour).
- Air changes: ca 10 air changes per hour.
- Photoperiod: 12 hours light/12 hours dark cycle (lights on from 7.00 AM to 7.00 PM).

IN-LIFE DATES:
- Start of study: 19/02/2007 (dose range finding test) and 28/02/2007 (main study).
- Termination of study (end of in-life phase): 05/03/2007.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2.5%, 5% and 10%.

No. of animals per dose:

5 females per dose.

Details on study design:

PRE-SCREEN TESTS:

- Experimental procedure: The dose range finding test was conducted in two parts. The first part comprised treatment of three groups of two animals each. Each group of two animals received one of the selected dose concentrations of 10%, 5% and 2.5% on the left ear. Due to absence of signs of irritation or systemic toxicity, the dose range finder test was extended by treatment of two groups of two animals each with concentrations of 20% and 50%. These concentrations were applied on the right ear of the animals that were previously treated with the 2.5% and 5% respectively in this dose range finder test. Each test substance formulation was administered on one ear per animal for three consecutive days. All animals were observed at least twice daily for signs of toxicity and their treated ear for signs of irritation. All findings were recorded.

- Irritation results: No abnormalities were observed in any of the animals after application of a 10%, 5% or 2.5% concentration on one of the ears. Erythema at the dorsum of the ear as a sign of irritation were observed on both animals treated with 25 µL of a 50% concentration of ETFBO on one of the ears.

- Systemic toxicity results: No abnormalities were observed in any of the animals after application of a 10%, 5% or 2.5% concentration on one of the ears. Blepharospasm was observed in both animals treated at a 20% concentration. Since these symptoms were not accompanied by body weight loss, the signs of toxicity after application of a 20% concentration were considered negligible. Body weight loss and blepharospasm as clear signs of toxicity were observed on both animals treated with 25 µL of a 50% concentration of ETFBO on one of the ears.

- Conclusion: No signs of excessive skin irritation were observed at any of the concentrations ETFBO used. Clear signs of toxicity were observed after three consecutive dermal applications of 25 µL of a 50% concentration of ETFBO. Based on these results, dermal application of 25 µL of a 20% is the highest concentration that meets the criteria for the main study to avoid systemic toxicity and excessive local skin irritation. Since both ears are treated in the main study and a total amount of 50 µL is applied (two times 25 µL), a 10% ETFBO concentration is the highest concentration selected for the main study.

MAIN STUDY

- Animal allocation: Twenty five mice were allocated to the various groups by computer randomisation on day -1. All animals assigned to this study were housed in one room during the study period. During the study, the animals were individually identified by a tail mark. Each cage was provided with a card showing the colour code, the animal identification number, the cage number, the group letter and the study number.

- Dose formulations:
* Test substance: Dose formulations in vehicle (4:1, v/v; acetone/olive oil) were freshly prepared on each day of dosing. The formulations were prepared on a volume/volume basis. After preparation of the required concentrations by addition of the vehicle to the test substance, the formulations were thoroughly mixed prior to dosing.
* Positive control substance: On each day of dosing, a fresh concentration of approximately 50% (w/v) of positive control substance in the vehicle, i.e. acetone/olive oil (4:1, v/v) was prepared.
All formulations were used for dosing within 1 hour of preparation.

- Experimental treatment: The main study comprised five groups; three test groups receiving the test substance ETFBO at dose levels of 2.5%, 5% and 10% (groups B, C and D, respectively), one control vehicle group (A), and one positive control group (E), each consisting of 5 females. On days 0, 1 and 2, the test substance at the selected concentrations of 2.5%, 5% and 10% and control substances were administered by pipetting 25 µL on the dorsum of both ears. Immediately after application, the formulation was dispersed over the ear by means of gently rubbing with the tip of the pipette. On day 5, all animals received an intravenous injection with 250 µL of phosphate-buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine in the tail vein. 3H-methyl thymidine was administered in a laboratory equipped for working with radio nuclides. Procedures followed were according to the regulations for working with radio nuclides. Five hours after the 3H-thymidine injection the animals were sacrificed by i.p. injection with euthanasate (sodium pentobarbital, approx. 200 mg/ml) and the draining auricular lymph nodes excised, pooled per animal in PBS and further processed to determine the 3H-thymidine incorporation.

- Observations and measurements:
* Clinical Observations: The general condition and behaviour of the animals were checked daily during the study. All findings including any abnormalities were recorded.
* Body weight: Body weights were determined at the start and at the end of the study.
* Necropsy: The animals were sacrificed on day 5 for excision of the auricular lymph nodes. The appearance of the lymph nodes and the ears were examined, but the animals were not subjected to full macroscopic examination.
* Assessment of 3H-thymidine incorporation in the auricular lymph nodes: Per animal, the paired auricular lymph nodes (ALN) were excised and were placed together in a vial containing 3 mL PBS. A single cell suspension was prepared by gently rubbing the nodes between the rough ends of microscope slides, followed by rinsing of the glass ends and the dish with PBS. The cell suspensions were washed twice (centrifugation 5 min, 300 g). The cell pellet obtained after the second wash was resuspended in 3 mL of 5% trichloroacetic acid (TCA) and left overnight (~18 hours) at 2-10°C. The precipitate was centrifuged (5 min, 450 g) and the supernatant discarded. The precipitate was resuspended in 3 mL Milli Q water. Subsequently 12 mL liquid scintillation cocktail (Ultima Gold, Packard) was added. The number of disintegrations per minute (DPM) was determined by counting for 10 minutes in a liquid scintillation counter (Wallac 1409).

- Criteria used to consider a positive response: 3H-thymidine incorporation was compared between the different treatment groups. Changes in 3H-thymidine incorporation in the test substance treatment groups were evaluated and expressed as stimulation index (SI). The SI was obtained by dividing the individual values of the 3H-thymidine incorporation, expressed as DPM (corrected for background), by the mean proliferation of the vehicle control group. The group-mean SI for vehicle-treated controls was set to one (1) by dividing the DPM-data of the individual vehicle-treated animals by the group-mean DPM-data. The decision process with regard to a positive response included a stimulation index >= 3 together with consideration of dose response and statistical analyses.

Positive control substance(s):

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

Statistics:

Statistical evaluation of the data (body weights and 3H-thymidine incorporation) was performed by analysis of variance followed by a Dunnett’s multiple comparison tests. In case of difference of variance between the different groups, log-transformation of the data before statistical evaluation can be considered. 3H-thymidine incorporation of treatment groups B, C and D were compared to vehicle-treated (A) animals. The validity of the model was tested by comparing the positive control (E) and the vehicle-treated (A) group. Probability values of p<0.05 were considered significant.

Results and discussion

Positive control results:

- Clinical Observations: Erythema at the dorsum of the ears was observed in all animals of the positive control group treated with a 50% concentration of HCA from day 2 to day 4.
- Body weight: No aberrant body weights and body weight gain were observed and no statistically significant differences were found between the vehicle and the positive control group.
- Necropsy: At necropsy, a small left auricular lymph node was noted in one positive control animal. No further abnormalities were observed in the other animals.
- Assessment of 3H-thymidine incorporation in the auricular lymph nodes: 3H-thymidine incorporation in the auricular lymph nodes of the positive control animal
which were treated with HCA, a known sensitizer, was statistically significant enhanced (p<0.001) in comparison with the vehicle controls. The calculated stimulation index of 8.7 supported this observation and demonstrated the sensitivity and validity of the model.

N.B. The concentration of HCA in acetone/olive oil (i.e. 50%) was higher than the one recommended in the current version of OECD 429 guideline (i.e. 25%). However, the recommendations that the positive control substance should give a SI > 3 whilst being not excessive (SI should not be above 20) was respected.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

12

Test group / Remarks:

Group treated with 2.5% ETFBO in vehicle

Results 2

Key result

Parameter:

SI

Value:

11

Test group / Remarks:

Group treated with 5% ETFBO in vehicle

Results 3

Key result

Parameter:

SI

Value:

16

Test group / Remarks:

Group treated with 10% ETFBO in vehicle

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA :
Significantly increased incorporation of 3H-thymidine was observed in the auricular lymph nodes of all ETBFO treated animals, achieving levels of statistical significance at all dose levels.

STIMULATION INDEX :
Expression of the 3H thymidine incorporation as a stimulation index compared to the vehicle control resulted in mean values of 12.0, 11.0 and 16.0 for groups treated with 2.5%, 5% and 10% ETFBO, respectively.

EC3 :
Not determinable because a clear dose response relationship was not present for the ETFBO concentrations of 2.5%, 5% and 10%.

CLINICAL OBSERVATIONS:
Erythema at the dorsum of the ears was observed in three out of five animals treated with a 10% concentration of ETFBO on days 4 and/or 5. No abnormalities were observed in the other two animals and in any of the animals treated with a 5% or 2.5% concentration of ETBFO or the vehicle. At necropsy, no abnormalities were observed in the animals treated with ETFBO.

BODY WEIGHTS:
No aberrant body weights and body weight gain were observed and no statistically significant differences were found between the vehicle group and the groups treated with ETFBO.

Any other information on results incl. tables

Mean values of auricular lymph node ³H-thymidine incorporation and body weight, as well as clinical signs recorded in each group:

Group	3H thymidine incorporation (DPM)	Stimulation Index (SI)	Clinical signs	Body weight
				Day 0	Day 5
Vehicle	1310.1	1.0	Ø	19.92	20.90
2.5% ETFBO	15703.4**	12.0	Ø	19.82	21.20
5% ETFBO	14443.8*	11.0	Ø	20.62	21.90
10% ETFBO	20965.4***	16.0	E (days 4-5) in 3 mice Ø in 2 mice	19.96	21.66
Positive control	11372.1***	8.7	E (days 2-4)	19.38	21.04

Statistical analysis (One Way Analysis of Variance): * p < 0.05; ** p < 0.01; *** p < 0.001

Ø = No abnormalities observed

E = Skin erythema (day numbers during which observation was seen)

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Stimulation indices of 12.0, 11.0 and 16.0 were calculated in response to a 2.5%, 5% and 10% ETFBO concentration, respectively. Since a stimulation index >= 3 is regarded as a positive response, the results indicated that ETFBO should be considered a skin sensitiser.

Executive summary:

The skin sensitisation potential was investigated in a GPL-compliant study performed according to OECD test guideline 429 (Local Lymph Node Assay, LLNA).

After a dose range finding test performed to determine the dose levels to be tested in the main study, three groups of 5 female mice were treated with increasing concentrations of ETFBO: 2.5%, 5% and 10% in vehicle (4:1, v/v; acetone/olive oil), with the vehicle alone and with a positive control substance (hexyl cinnamic aldehyde, HCA). Administration of dosing formulations occurred on days 0, 1 and 2 by pipetting 25 µl on the dorsum of both ears. On day 5, all animals received an intravenous injection of ³H-thymidine. Five hours later, the ³H-thymidine incorporation in the draining auricular lymph nodes was determined and expressed as a stimulation index (SI). A SI >=3 is regarded as a positive response. The obtained results were compared with those of the control group which was treated with the vehicle alone. The positive control group treated with HCA, a known sensitizer, served as a reliability check.

In the positive control group after three consecutive applications, erythema were seen at the dorsum of the ears between days 2 to 4, and ³H-thymidine incorporation in the auricular lymph nodes was significantly enhanced in comparison with the vehicle controls. The calculated stimulation index of 8.7 supported this observation and demonstrated the sensitivity and validity of the model. Erythema at the dorsum of the ears was observed in some animals treated with a 10% ETFBO concentration on days 4 and/or 5. No abnormalities were observed in the other two animals and in any of the animals treated with a 5% or 2.5% concentration of ETBFO or the vehicle. ³H-thymidine incorporation in the auricular lymph nodes was significant enhanced at all three ETFBO dose levels in comparison with the vehicle controls. Stimulation indices of 12.0, 11.0 and 16.0 were calculated in response to a 2.5%, 5% and 10% ETFBO concentration respectively. EC3 value was not determinable because a clear dose response relationship was not present.

Since the stimulation indices are >=3, ETFBO has to be considered as a skin sensitizer and according to the GHS criteria, has to be classified in Category 1 for skin sensitisation. Because it was not possible to determine an EC3 value, further subcategorization is not possible.