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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:
07 October 2015 to 02 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dichlorobenzoyl chloride
EC Number:
220-813-6
EC Name:
3,5-dichlorobenzoyl chloride
Cas Number:
2905-62-6
Molecular formula:
C7H3Cl3O
IUPAC Name:
3,5-dichlorobenzoyl chloride
Test material form:
other: solid (unspecified)
Details on test material:
- Appearance: White solid
- Storage conditions of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/J strain, inbred, SPF-Quality
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 20.2 to 23.8 g (body weight variation was within ± 20 % of the sex mean)
- Housing: During acclimation animals were group housed in labelled cages (height 18 cm) containing sterilised sawdust as bedding material; paper and shelters were supplied as cage-enrichment. After acclimation animals were group housed in cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % relative
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: No data

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 2, 5 and 10 % test material (w/w)
No. of animals per dose:
5 per dose
Details on study design:
PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test material concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2) and/or an increase in ear thickness < 25 %) and/or is the highest possible concentration that can technically be applied.
Two test material concentrations were tested; 10 and 25 % concentrations in the chosen vehicle.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labelled cages. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

Both animals treated at a concentration of 25 % showed very slight erythema between Days 3 and 5. Scaliness was noted for these animals on Day 6. Variations in ear thickness during the observation period were more than 25 % from Day 1 pre-dose values.
Very slight erythema (Day 4) was observed in the animals treated at a concentration of 10 %. Variations in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values for these animals. No signs of systemic toxicity were observed in these animals.
Based on these results, the highest test material concentration selected for the main study was a 10 % concentration.

MAIN STUDY
Three groups of five animals were treated with one test material concentration per group. The highest test material concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

INDUCTION - DAYS 1, 2 AND 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals.

EXCISION OF THE NODES - DAY 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of ³H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20 %. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING FOR RADIOACTIVITY - DAY 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - DAY 7
Precipitates were recovered by centrifugation, re-suspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS
-Mortality/Viability: Twice daily
- Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy)
- Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing)
- Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition, a description of all other (local) effects was recorded.
Grading Irritation Reactions (erythema and eschar formation):
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth): 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4
- Necropsy: No necropsy for gross macroscopic examination was performed
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A reliability test with three concentrations of α-hexylcinnamaldehyde in acetone/olive oil (4:1 v/v) was performed not more than 6 months previously, using the same materials, animal supplier, animal strain and essential procedures as for the main test. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.
The dose levels were 0, 5, 10 and 25 % in acetone:olive oil (4:1 v/v)
The SI values calculated for the material concentrations 5, 10 and 25 % were 1.7, 3.0 and 9.1 respectively. An EC3 value of 10 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %. The results of the 6 monthly HCA reliability checks of the recent years were 16.5, 14.5, 13.4, 14.1, 17.3 and 9.8 %.
Based on the results, it was concluded that the LLNA as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
12.6
Test group / Remarks:
Test material concentration: 2 %
Key result
Parameter:
SI
Value:
17.9
Test group / Remarks:
Test material concentration: 5 %
Key result
Parameter:
SI
Value:
21.5
Test group / Remarks:
Test material concentration: 10 %
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test material concentrations 2, 5 and 10 % were 12 894, 18 366 and 22 095 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1027 DPM.

Any other information on results incl. tables

Skin Reactions / Irritation

The slight irritation of the ears as shown by three animals treated at 10 % was considered not to have a toxicologically significant effect on the activity of the nodes.

 

Systemic Toxicity

No mortality occurred. Three animals treated at 10 % showed piloerection on Day 3. This was considered to have no effect on the activity of the nodes since it was noted on one day only. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area

Most auricular lymph nodes of the animals of the experimental groups were considered enlarged in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Result

These results show that the test material elicits a SI ≥ 3. The data showed a dose-response and the EC3 value (the estimated test material concentration that will give a SI =3) was established to be between >0 and 2 %.

Table 1: Relative Size of Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Group

Test Material

(% w/w)

Animal Number

Size nodes*

DPM/animal

Mean DPM ± SEM

Mean SI ± SEM

Left

Right

1

0

1

n

n

758

1027 ± 174

1.0 ± 0.2

2

n

n

908

3

n

n

1557

4

n

n

1295

5

n

n

616

2

2

6

+

n

7812

12 894 ± 2123

12.6 ± 3.0

7

+

+

7965

8

+

+

17 949

9

+

+

16 421

10

+

++

14 324

3

5

11

++

++

12 144

18 366 ± 4150

17.9 ± 5.1

12

+

+

5162

13

++

++

24 545

14

++

++

23 333

15

++

++

26 647

4

10

16

+

+

20 981

22 095 ± 3110

21.5 ± 4.7

17

++

++

30 649

18

++

++

11 449

19

++

++

23 158

20

++

++

24 237

*Relative size auricular lymph nodes (-, -- or ---: degree of reduction; +,++ or +++: degree of enlargement; n: considered to be normal).

DPM = Disintegrations per minute

SEM = Standard Error of the Mean

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study, the test material elicited a SI ≥ 3. The test material is therefore considered to be a sensitiser.
Executive summary:

The potential for the test material to cause skin sensitisation was investigated in the mouse in a Local Lymph Node Assay (LLNA) carried out in accordance with the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600 under GLP conditions.

Test material concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 2, 5 or 10 % w/w in acetone/olive oil (4:1 v/v) on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone.

Three days after the last exposure, all animals were injected with ³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Slight irritation of the ears was shown by three animals treated at 10 % and was considered not to have a toxicologically significant effect on the activity of the nodes.

Most auricular lymph nodes of the animals of the experimental groups were considered enlarged in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test material concentrations 2, 5 and 10 % were 12 894, 18 366 and 22 095 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1027 DPM. The SI values calculated for the material concentrations 2, 5 and 10 % were 12.6, 17.9 and 21.5, respectively.

These results show that the test material elicits a SI ≥ 3. The data showed a dose-response and the EC3 value (the estimated test material concentration that will give a SI =3) was established to be between >0 and 2 %.

Under the conditions of this study, the test material is considered to be a sensitiser.

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