Hexachloroacetone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Study - DRAFT

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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

Test material information

Constituent 1

Reference substance name:

Hexachloroacetone

EC Number:

204-129-5

EC Name:

Hexachloroacetone

Cas Number:

116-16-5

Molecular formula:

C3Cl6O

IUPAC Name:

hexachloropropan-2-one

Test material form:

other: colourless liquid

Details on test material:

Purity: 99.2% (GC)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
Sex: female (nulliparous and non-pregnant)
Age at the beginning of the study: 8-9 weeks
Number of animals: 5 mice / group
5 mice / prescreen test
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals are bred for experimental purposes.
Full barrier in an air-conditioned room
Temperature: 22 ± 3 °C
Relative humidity: 55 ± 10%
Artificial light, sequence being 12 hours light, 12 hours dark
Air change: at least 10 x / hour
Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main study: lot no. 1239)
Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (prescreen test and main study: lot no. 02102150227)
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
Adequate acclimatisation period (at least five days) under laboratory conditions
The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).
Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.
Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).
Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25%, 50% (each diluted with AOO 4:1, v/v) and 100% (undiluted test item)

No. of animals per dose:

5 mice / group
5 mice / prescreen test

Details on study design:

Topical Application
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application.
Topical applications were performed once daily over three consecutive days.
Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted to a working concentration of 80µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing the thickness of the ears of all animals was measured for a third time. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: P-Phenylenediamine

Statistics:

EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Results and discussion

Positive control results:

Stimulation Index of P-Phenylenediamine: 13,2 +/- 2.2

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Each of the three tested concentrations of the test item reached the stimulation index of3. The stimulation index at a concentration of 25% was 27.3 The stimulation index at a concentration of 50% was 33.1 The stimulation index at a concentration of 100% was 19.8

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

Any other information on results incl. tables

Directly prior to the first application, approximately 48 hours after the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured.

The means of the ear thickness per group showed no relevant difference compared to the negative control.

The mean ear thickness on	day 1	day 3	day 6
for the 25% test group was	0.17 mm	0.18 mm	0.18 mm
for the 50% test group was	0.18 mm	0.18 mm	0.18 mm
for the 100% test group was	0.17 mm	0.18 mm	0.18 mm
for the negative control group was	0.17 mm	0.17 mm	0.17 mm

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

Each of the tested concentrations exceeded the stimulation index of 3. The EC3 value could not be calculated due to the lack of a dose-response relationship of the data obtained.
Consequently, according to OECD 429 the test item HEXACHLOROACETONE is expected to be a dermal sensitiser. Therefore, according to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item HEXACHLOROACETONE has obligatory labelling requirement for skin sensitisation and is classified into Category 1.

Executive summary:

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 25% (v/v), 50% (v/v) , each diluted with AOO 4:1, (v/v) and 100% (undiluted test item).

At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed.

Species/strain:                     Mice, CBA/CaOlaHsd

Number of animals:              20/main test

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

Summary Results

Eachof the three tested concentrations of the test item reached the stimulation index of3.

The stimulation index at a concentrationof        25%    was     27.3

The stimulation index at a concentrationof        50%    was     33.1

The stimulation index at a concentrationof        100%  was     19.8

Classification in sub-category 1A or 1B cannot be made as the EC3 value could not be calculated due to the lack of dose-response relationship of the data obtained. The means of the ear thickness per group showed no relevant difference compared to the negative control.

The mean ear thickness on		day 1		day 3		day 6
for the 25% test group was		0.17 mm		0.18 mm		0.18 mm
for the 50% test group was		0.18 mm		0.18 mm		0.18 mm
for the 100% test group was		0.17 mm		0.18 mm		0.18 mm
for the negative control group was		0.17 mm		0.17 mm		0.17 mm

 

Conclusion

Each of the tested concentrations exceeded the stimulation index of 3. The EC3 value could not be calculated dueto the lack of a dose-response relationship of the data obtained.

Consequently, according to OECD 429 the test item HEXACHLOROACETONE is expected to be a dermal sensitiser. Therefore, according to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item HEXACHLOROACETONE has obligatory labelling requirement for skin sensitisation and is classified into Category 1.