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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2018 to 27 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 429 Skin Sensitization: Local Lymph Node Assay (22 July 2010)
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.42., “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
Deviations:
yes
Remarks:
See "any other information" for details
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Chemical name: α,α’-Dichloro-p-xylene
CAS number: 623-25-6
Batch/Lot Number: EWP6O
Description: White-Pale yellow green, Solid
Purity*: >98.0% (GC)
Expiry date: 17 August 2018 / 30 October 2019
Storage conditions: Controlled room temperature (15-25 °C, ≤ 70 RH%), protected from humidity (tight closed container), protected from light
Safety precautions: Enhanced safety precautions were applied considering the supplied safety data sheet to assure personnel health and safety. Fatal if inhaled!
Specific details on test material used for the study:
No further details specified in the study report.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: Specific Pathogen Free (SPF) at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline 429, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals (main study): 12 weeks old (age-matched, within one week)
Body weight range (main study): 20.4 – 23.8 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 35 days
Note: In the Preliminary Experiment I, mice of 11 weeks of age (19.6-19.8 grams) were used. In the Preliminary Experiment II mice of 12 weeks of age (19.8-20.7 grams) were used. In the Preliminary Experiment III mice of 13 weeks of age (20.0-21.9 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 16.7 – 27.3°C
Relative humidity: 22 - 86 %
Ventilation: 15-20 air exchanges/hour
The minimum /maximum values of temperature and relative humidity were recorded twice every day during the acclimatization and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 382 24962, Expiry date: April 2018, produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60180640040101, Expiry date: 05 March 2019 and Batch Number: 60172780020101, Expiry date: 05 October 2018) Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Citoxlab Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
The Preliminary Irritation/Toxicity Test I was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 25% (w/v) and 10% (w/v) in DMF.
Preliminary Irritation/Toxicity Test II using three doses (2 animals/dose) at test item concentrations of 5% (w/v) and 2% (w/v) and 1%(w/v) in DMF.
Preliminary Irritation/Toxicity Test III using three doses (2 animals/dose) at test item concentrations of 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v) in DMF.
Main Study using three doses (4 animals/dose) at test item concentrations of 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v) in DMF.
No. of animals per dose:
2 animals / group - preliminiart studies
4 animals / group - main study
Details on study design:
ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test I was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 25% (w/v) and 10% (w/v) in DMF. Based on the observed ear swelling and clinical observations an additional test was performed. Preliminary Irritation/Toxicity Test II using three doses (2 animals/dose) at test item concentrations of 5% (w/v) and 2% (w/v) and 1%(w/v) in DMF. Based on the observed ear swelling an additional test was performed. Preliminary Irritation/Toxicity Test III using three doses (2 animals/dose) at test item concentrations of 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v) in DMF.
The preliminary experiments were conducted in a similar experimental manner to the main study, but were terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 25 % (w/v).
In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored using Table 2 [3, 4]. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

Clinical observations
During the Preliminary Irritation / Toxicity Tests, no mortality was observed.
In the Preliminary Experiment I minimal amount of test item precipitate was observed on the ears of both animals in 25% (w/v) dose group on Days 1-3. Hyperactivity and intermittent tremors were observed on Day 3 and rigid ears were observed on Days 5-6 for both animals of the 25%(w/v) dose group. Hyperactivity was observed on Day 3 for one animal of the 10% (w/v) dose group.
In the Preliminary Experiment II slight erythema (score 1) was observed on Days 4-5 for both animals of the 5%(w/v) dose group.
The revealing ear punch weights were out of the historical control range for both animals of the 25% (w/v), 10% (w/v), 5% (w/v), 2% (w/v) dose groups and for one animal of the 1% (w/v) dose group, the increase was above the limit of positive (≥25%) for both animals of the 25% (w/v), 10% (w/v) dose groups and for one animal of the 5%(w/v) dose group.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in all animals of the 25% (w/v), 10% (w/v), 5% (w/v), 2% (w/v) and 1% (w/v) dose groups. They were normal in all animals of the 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v) dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 25%, 10%, 5%, 2% and 1% (w/v) doses clearly exceeded the acceptable limit; therefore, these concentrations were excluded from the examined concentration series of the main test. Slightly increased ear thickness values were seen for one animal (left ear) at 0.5% (w/v) concentration, thus it was selected as top dose for the main test. Additionally, ear thickness of the experimental animals in the main test was measured by using a thickness gauge on Days 1 and 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals are humanely killed.

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of 0.1 g.

Measurement of Ear Thickness
The ear thickness values of the experimental animals were determined by using a thickness gauge on Day 1 (pre-dose) and Day 6 in the study.
Additional quantification of the ear thickness was also performed in the study by ear punch weight determination on Day 6 after the animals were euthanized.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile Phosphate Buffered Saline (PBS) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before any incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently resuspended and 3 mL of 5 % (w/v) TCA solution added to the tubes for precipitation of macromolecules.
After overnight incubation (approximately 18 hours) at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
11.2
Test group / Remarks:
0.5% (w/v)
Key result
Parameter:
SI
Value:
8.9
Test group / Remarks:
0.25% (w/v)
Key result
Parameter:
SI
Value:
6.9
Test group / Remarks:
0.1% (w/v)
Cellular proliferation data / Observations:
CLINICAL OBSERVATION
No mortality or systemic clinical signs were observed during the main study. Minimal amount of test item precipitate was observed on the ears of one animal of the 0.5% (w/v) dose group on Day 1. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
Marked body weight loss (≥5%) was observed for two animals of the 0.1% (w/v) dose group and for one animal of the 0.25(w/v) dose group and for one animal of the positive control group. However, the mean body weight of these groups were in the acceptable range and these changes were considered as individual variability. No treatment related effects were observed on the mean body weight changes in the main study.

EAR THICKNESS MEASUREMENT
The ear thickness values and the biopsy weights were within the acceptable range for all the animals in the test item treated groups and in the negative control group.
The detected ear thickness values indicated local irritation (≥ 25 %) for all animals of the positive control group on Day 6. The revealing ear punch weights were below the limit of positive (≥25%). Occasionally local irritation is seen with the positive control substance, it is not considered to affect the study interpretation.
This fact was considered not to adversely affect the results or integrity of the study.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group. Larger than normal lymph nodes were observed in the 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v) test item treated dose groups and in the positive control group.
The stimulation index values were 11.2, 8.9 and 6.9 at concentrations of 0.5% (w/v), 0.25% (w/v) and 0.1 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
The test item was solid, which was formulated in DMF. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations used in the main study, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that α,α’-Dichloro-p-xylene is a strong sensitizer. The size of lymph nodes was in good correlation with this conclusion.
Based on the observed results the test item is classified as Category 1 (sub-category 1A) according to the GHS or CLP.
The extrapolated EC3 value of α,α’-Dichloro-p-xylene is 0.02% (w/v).

RELIABILITY OF THE TEST
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay [1]. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (DMF) using CBA/CaOlaHsd mice.
No mortality cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 7.5) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

Any other information on results incl. tables

Individual Body Weights for all Animals with Group Means

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

5915

5919

5922

5925

 

1

2

3

4

Negative (vehicle) control DMF

 

 

Mean

23.3

21.7

21.8

20.9

21.9

23.9

21.6

21.7

20.9

22.0

2.6

-0.5

-0.5

0.0

0.4

5917

5926

5929

5921

 

5

6

7

8

α,α'-Dichloro-p-xylene

0.5 (w/v) %

in DMF

 

Mean

23.3

21.7

21.7

20.9

21.9

22.9

22.1

20.8

20.3

21.5

-1.7

1.8

-4.1

-2.9

-1.7

5924

5930

5918

5928

 

9

10

11

12

 

α,α'-Dichloro-p-xylene

0.25 (w/v) %

in DMF

 

Mean

23.8

22.0

21.7

20.9

22.1

22.8

20.5

22.4

20.9

21.7

-4.2

-6.8

3.2

0.0

-1.9

5927

5916

5923

5920

 

13

14

15

16

αα'-Dichloro-p-xylene

0.1 (w/v) %

in DMF

 

Mean

23.6

21.9

21.7

20.4

21.9

21.6

22.8

20.4

21.0

21.5

-8.5

4.1

-6.0

2.9

-1.9

5933

5931

5932

5934

 

17

18

19

20

Positive control

25 (w/v) % HCA

in DMF

 

Mean

22.9

22.4

21.6

21.1

22.0

21.1

22.2

21.3

21.3

21.5

-7.9

-0.9

-1.4

0.9

-2.3

Notes:

*: Terminal body weights were measured on Day 6

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DMP / group

DPM

Number of lymph nodes

DPN

Stimulation Index

Background

(5% (w/v) TCA)

49

39

-

-

-

-

Negative control

(DMF)

4386

4342.0

8

542.8

1.0

α,α'-Dichloro-p-xylene

0.5 (w/v) %

in DMF

48665

48621.0

8

6077.6

11.2

α,α'-Dichloro-p-xylene

0.25 (w/v) %

in DMF

38884

38840.0

8

4855.0

8.9

α,α'-Dichloro-p-xylene

0.1 (w/v) %

in DMF

29850

29806.0

8

3725.8

6.9

Positive control

(25% (w/v) HCA in DMF)

32684

3264.0

8

4080.0

7.5

 

Results of the Preliminary Irritation / Toxicity Test I

Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test I)

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

5575

5576

1

2

25% (w/v)

25% (w/v)

Mean

19.8

19.6

19.7

17.9

19.6

18.8

-9.6

0.0

-4.8

5577

5574

3

4

10% (w/v)

10% (w/v)

Mean

19.7

19.6

19.7

19.1

19.4

19.3

-3.0

-1.0

-2.0

Notes:

1. *: Terminal body weights were measured on Day 6.

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Irritation/Toxicity Test I)

Animal Number

Identity                Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 3 (mm)*

Ear Thickness on Day 6 (mm)*

Biopsy weight** on Day 6 (mg)

Right

Left

Right

Left

Right

Left

5575

5576

5577

5574

1

2

3

4

25% (w/v)

25% (w/v)

10% (w/v)

10% (w/v)

0.21

0.21

0.21

0.21

0.20

0.21

0.21

0.21

0.38

0.33

0.31

0.28

0.35

0.37

0.31

0.29

0.54

0.53

0.42

0.35

0.50

0.36

0.38

0.41

38.65

33.57

29.39

31.70

Notes:

1. *: In case of ear thickness values, irritant response is considered when result is ≥25% above the Day 1 value.

2. **: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).

 

Summarized Clinical Observations (Preliminary Irritation/Toxicity Test I)

Period

Group

Identity No.

Animal No.

Clinical observations

DAY 1

25% (w/v)

1

5575

Before treatment: symptom-free, ES: 0

After treatment: symptom-free**, ES: 0

2

5576

Before treatment: symptom-free, ES: 0

After treatment: symptom-free**, ES: 0

10% (w/v/)

3

5577

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5574

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 2

25% (w/v)

1

5575

Before treatment: symptom-free, ES: 0

After treatment: symptom-free**, ES: 0

2

5576

Before treatment: symptom-free, ES: 0

After treatment: symptom-free**, ES: 0

10% (w/v/)

3

5577

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5574

Before treatment: symptom-free, ES: 0

After treatment: symptom-free ES: 0

DAY 3

25% (w/v)

1

5575

Before treatment: symptom-free, ES: 0

After treatment: hyperactivity, intermittent tremors**, ES: 0

2

5576

Before treatment: symptom-free, ES: 0

After treatment: hyperactivity, intermittent tremors**, ES: 0

10% (w/v/)

3

5577

Before treatment: symptom-free, ES: 0

After treatment: symptom-free ES: 0

4

5574

Before treatment: symptom-free, ES: 0

After treatment: symptom-free ES: 0

DAY 4

25% (w/v)

1

5575

Symptom-free**, ES: 0

2

5576

Symptom-free**, ES: 0

10% (w/v/)

3

5577

Symptom-free, ES: 0

4

5574

Symptom-free, ES: 0

DAY 5

25% (w/v)

1

5575

Rigid ears, ES: 0

2

5576

Rigid ears, ES: 0

10% (w/v/)

3

5577

Symptom-free, ES: 0

4

5574

Symptom-free, ES: 0

DAY 6

25% (w/v)

1

5575

Rigid ears, ES: 0

2

5576

Rigid ears, ES: 0

10% (w/v/)

3

5577

Symptom-free, ES: 0

4

5574

Symptom-free, ES: 0

Notes:

1. The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. **: minimal amount of test item precipitate

 

Results of the Preliminary Irritation / Toxicity Test II

Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test II)

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

5645

5647

1

2

5% (w/v)

5% (w/v)

Mean

20.2

19.8

20.0

19.3

19.0

19.2

-4.5

-4.0

-4.2

5643

5646

3

4

2% (w/v)

2% (w/v)

Mean

201.

20.7

20.4

19.8

21.3

20.6

-1.5

2.9

0.7

5648

5644

5

6

1% (w/v)

1% (w/v)

Mean

20.3

20.5

20.4

20.6

19.1

19.9

1.5

-6.8

-2.7

Notes:

1. *: Terminal body weights were measured on Day 6.

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Irritation/Toxicity Test II)

Animal Number

Identity                Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 3 (mm)*

Ear Thickness on Day 6 (mm)*

Biopsy weight** on Day 6 (mg)

Right

Left

Right

Left

Right

Left

5645

5647

5643

5646

5648

5644

1

2

3

4

5

6

5% (w/v)

5% (w/v)

2% (w/v)

2% (w/v)

1% (w/v)

1% (w/v)

0.22

0.21

0.21

0.20

0.22

0.21

0.22

0.20

0.22

0.21

0.22

0.22

0.32

0.31

0.24

0.26

0.24

0.24

0.29

0.29

0.23

0.26

0.23

0.25

0.36

0.38

0.28

0.31

0.30

0.27

0.34

0.44

0.28

0.30

0.32

0.29

27.10

31.97

26.86

25.48

20.06

23.59

Notes:

1. *: In case of ear thickness values, irritant response is considered when result is ≥25% above the Day 1 value.

2. **: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).

 

Summarized Clinical Observations (Preliminary Irritation/Toxicity Test II)

Period

Group

Identity No.

Animal No.

Clinical observations

DAY 1

5% (w/v)

1

5645

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5647

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2% (w/v)

3

5643

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5646

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

1% (w/v/)

5

5648

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

6

5644

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 2

5% (w/v)

1

5645

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5647

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2% (w/v)

3

5643

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5646

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

1% (w/v/)

5

5648

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

6

5644

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 3

5% (w/v)

1

5645

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5647

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2% (w/v)

3

5643

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5646

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

1% (w/v/)

5

5648

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

6

5644

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 4

5% (w/v)

1

5645

ES: 1

2

5647

ES: 1

2% (w/v)

3

5643

Symptom-free, ES: 0

4

5646

Symptom-free, ES: 0

1% (w/v/)

5

5648

Symptom-free, ES: 0

6

5644

Symptom-free, ES: 0

DAY 5

5% (w/v)

1

5645

ES: 1

2

5647

ES: 1

2% (w/v)

3

5643

Symptom-free, ES: 0

4

5646

Symptom-free, ES: 0

1% (w/v/)

5

5648

Symptom-free, ES: 0

6

5644

Symptom-free, ES: 0

DAY 6

5% (w/v)

1

5645

Symptom-free, ES: 0

2

5647

Symptom-free, ES: 0

2% (w/v)

3

5643

Symptom-free, ES: 0

4

5646

Symptom-free, ES: 0

1% (w/v/)

5

5648

Symptom-free, ES: 0

6

5644

Symptom-free, ES: 0

Notes:

1. The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

 

Results of the Preliminary Irritation / Toxicity Test III

Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test III)

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

5657

5659

1

2

0.5% (w/v)

0.5% (w/v)

Mean

20.8

21.9

21.4

19.3

20.2

19.8

-7.2

-7.8

-7.5

5660

5662

3

4

0.25% (w/v)

0.25% (w/v)

Mean

21.0

21.6

21.3

20.5

22.5

21.5

-2.4

4.2

0.9

5658

5661

5

6

0.1% (w/v)

0.1% (w/v)

Mean

20.0

21.7

20.9

18.9

21.2

20.1

-5.5

-2.3

-3.9

Notes:

1. *: Terminal body weights were measured on Day 6.

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Irritation/Toxicity Test III)

Animal Number

Identity                Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 3 (mm)*

Ear Thickness on Day 6 (mm)*

Biopsy weight** on Day 6 (mg)

Right

Left

Right

Left

Right

Left

5657

5659

5660

5662

5658

5661

1

2

3

4

5

6

0.5% (w/v)

0.5% (w/v)

0.25% (w/v)

0.25% (w/v)

0.1% (w/v)

0.1% (w/v)

0.22

0.22

0.22

0.22

0.22

0.22

0.22

0.23

0.22

0.22

0.23

0.22

0.24

0.23

0.23

0.23

0.24

0.23

0.26

0.22

0.24

0.23

0.23

0.23

0.23

0.23

0.22

0.23

0.22

0.22

0.25

0.24

0.22

0.22

0.23

0.22

15.71

16.04

13.86

16.69

13.54

14.10

Notes:

1. *: In case of ear thickness values, irritant response is considered when result is ≥25% above the Day 1 value.

2. **: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).

 

Summarized Clinical Observations (Preliminary Irritation/Toxicity Test III)

Period

Group

Identity No.

Animal No.

Clinical observations

DAY 1

0.5% (w/v)

1

5657

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5659

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

0.25% (w/v)

3

5660

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5662

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

0.1% (w/v/)

5

5658

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

6

5661

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 2

0.5% (w/v)

1

5657

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5659

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

0.25% (w/v)

3

5660

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5662

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

0.1% (w/v/)

5

5658

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

6

5661

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 3

0.5% (w/v)

1

5657

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

5659

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

0.25% (w/v)

3

5660

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

5662

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

0.1% (w/v/)

5

5658

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

6

5661

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 4

0.5% (w/v)

1

5657

Symptom-free, ES: 0

2

5659

Symptom-free, ES: 0

0.25% (w/v)

3

5660

Symptom-free, ES: 0

4

5662

Symptom-free, ES: 0

0.1% (w/v/)

5

5658

Symptom-free, ES: 0

6

5661

Symptom-free, ES: 0

DAY 5

0.5% (w/v)

1

5657

Symptom-free, ES: 0

2

5659

Symptom-free, ES: 0

0.25% (w/v)

3

5660

Symptom-free, ES: 0

4

5662

Symptom-free, ES: 0

0.1% (w/v/)

5

5658

Symptom-free, ES: 0

6

5661

Symptom-free, ES: 0

DAY 6

0.5% (w/v)

1

5657

Symptom-free, ES: 0

2

5659

Symptom-free, ES: 0

0.25% (w/v)

3

5660

Symptom-free, ES: 0

4

5662

Symptom-free, ES: 0

0.1% (w/v/)

5

5658

Symptom-free, ES: 0

6

5661

Symptom-free, ES: 0

Notes:

1. The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

 

Summarized Clinical Observations and Individual Ear Thickness Values

Individual Ear Thickness for all Animals

Animal Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 6 (mm)*

Biopsy weight on Day 6**

Right

Left

Right

Left

(mg)

5915

Negative (vehicle) control

DMF

0.22

0.21

0.21

0.22

0.22

0.22

0.22

0.22

0.23

0.22

0.21

0.23

0.22

0.22

0.22

0.22

15.7

15.1

16.1

15.9

5919

5922

5925

5917

α,α'-Dichloro-p-xylene

0.5 (w/v) %

in DMF

0.21

0.22

0.21

0.22

0.21

0.22

0.20

0.22

0.22

0.22

0.25

0.22

0.22

0.22

0.23

0.24

18.6

17.1

18.6

17.3

5926

5929

5921

5924

α,α'-Dichloro-p-xylene

0.25 (w/v) %

in DMF

0.21

0.22

0.21

0.21

0.21

0.21

0.21

0.21

0.22

0.22

0.23

0.23

0.21

0.21

0.23

0.22

16.8

17.6

18.2

16.2

5930

5918

5928

5927

α,α'-Dichloro-p-xylene

0.1 (w/v) %

in DMF

0.22

0.22

0.21

0.21

0.22

0.22

0.21

0.22

0.22

0.22

0.21

0.22

0.22

0.22

0.21

0.22

15.9

15.9

15.2

16.2

5916

5923

5920

5933

Positive control

25 (w/v/) % HCA

in DMF

0.22

0.22

0.22

0.23

0.22

0.22

0.21

0.22

0.38

0.37

0.38

0.36

0.36

0.38

0.35

0.33

25.6

23.5

24.5

22.9

5931

5932

5934

Notes:

1. *: In case of ear thickness values, irritant response is considered when result is ≥25% above the Day 1 value.

2. **: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).

 

Summarized Clinical Observations

Group

Identity No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(DMF)

1

 

2

 

3

 

4

 

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

α,α'-Dichloro-p-xylene

0.5 (w/v) % in DMF

5

 

6

 

7

 

8

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

α,α'-Dichloro-p-xylene

0.25 (w/v) % in DMF

9

 

10

 

11

 

12

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

α,α'-Dichloro-p-xylene

0.1 (w/v) % in DMF

13

 

14

 

15

 

16

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Positive control (25% (w/v/) HCA in DMF)

17

 

18

 

19

 

20

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Notes:

1. BT: before treatment. AT: after treatment

2. **: minimal amount of test item precipitate

 

Historical Control Data

Historical Control data of the Positive and negative Controls for CBA/CaOlaHsd mice (2014-2018)

 

Vehicles

Acetone : Olive oil 4:1 (AOO)

1% Pluronic PE9200 in water

(1% Plu)

DPN values

SI value

DPN values

SI value

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

472.7

3851.3

9.0

198.7

1988.1

11.2

Range:           min

           max

35.8

1990.1

890.3

10336.0

3.3

20.2

23.0

680.8

154.0

6755.8

3.0

33.6

Number of cases

92

88

86

234

226

218

 

 

 

Vehicles

N,N-Dimethlyformamide (DMF)

Dimethyl sulfoxide (DMSO)

DPN values

SI value

DPN values

SI value

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

256.1

2738.9

11.3

466.0

3014.4

7.2

Range:           min

           max

62.0

649.6

1201.3

5817.9

4.9

21.3

218.3

934.6

1461.3

4877.5

3.1

14.5

Number of cases

68

68

68

22

22

21

 

 

Vehicles

Propylene glycol (PG)

Methyl ethyl ketone (MEK)

DPN values

SI value

DPN values

SI value

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

245.1

2278.6

9.4

264.5

4129.5

16.9

Range:           min

           max

63.3

506.0

817.3

4978.0

5.8

14.4

80.5

516.2

1562.5

8682.5

8.8

36.3

Number of cases

18

18

18

18

19

19

HCA 25% = alpha-Hexylcimmaldehyde 25 % (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, under the conditions of the present assay, α,α’-Dichloro-p-xylene, tested in a suitable vehicle, was shown to have a strong sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of α,α’-Dichloro-p-xylene is 0.02% (w/v).
The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1A).
Executive summary:

The aim of this study was to determine the skin sensitisation potential of α,α’-Dichloro-p-xylene following dermal exposure in mice. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, in accordance to the regulatory guidelines for animal welfare.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the best vehicle for the test item was N,N-dimethylformamide (DMF). The 25% (w/v) test item formulation was chosen as the highest suitable concentration for the test. The formulations appeared to be a solution by visual examination.

 

Preliminary Irritation/Toxicity Tests were performed in CBA/CaOlaHsd mice using two or three doses (2 animals/dose) at 25%, 10%, 5%, 2%, 1%, 0.5%, 0.25% and 0.1% (w/v) in DMF. Based on local irritation effects in the preliminary tests, 0.5% (w/v) dose was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received α,α’-Dichloro-p-xylene (formulated in DMF) at 0.5% (w/v), 0.25% (w/v) and 0.1 % (w/v) concentrations respectively,

- the negative control group received the vehicle (DMF) only,

- the positive control group received 25 % (w/v) HCA (dissolved in DMF).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

No mortality or systemic clinical signs were observed during the main study. Minimal amount of test item precipitate was observed on the ears of one animal of the 0.5% (w/v) dose group on Day 1. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the main study. The ear thickness values and the biopsy weights were within the acceptable range for all the animals in the test item treated groups.

 

The stimulation index values were 11.2, 8.9 and 6.9 at concentrations of 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v), respectively.

 

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, α,α’-Dichloro-p-xylene, tested in a suitable vehicle, was shown to have a strong sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of α,α’-Dichloro-p-xylene is 0.02% (w/v).

 

The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1A).