Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2003 to 26 May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: HSY-2701
Description: Red powder
Batch: 03A-20
Purity: >99.9 %
Test substance storage: At room temperature protected from light
Stability under storage conditions: Stable
Expiry date: 07 August 2003 (6 months from the date of receipt).

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 19 - 24 g
- Housing: Individual housing in labelled Macrolon caged (type I; height 12.5 cm) containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany).
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany)
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least five days, housed in polycarbonate cages (Macrolon II type; height 15 cm).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.8 to 23.0 °C
- Humidity (%): 35 to 82 %
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark per day.

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
1, 10 and 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Homogeneity was obtained to visually acceptatble levels.
- Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100 % (undiluted), 50%, 25 %, 10 %, 5 %, 2.5%, 1 % and were needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to theose used during days 1 to 3 of the main study unless otherwise specified. Four young adult animals were selected (approx. 10 weeks old). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleared of residual test substance with water and the irritation was assessed. No necrospy was performed and no body weights were determined after termination.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: If the results indicated a SI ≥3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-4494, February 1999).

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of four animals were treated with three test substance concentrations. The vehicle control group was used from the simultaneously treated NOTOX Project 371216. The animals were allocated into the following groups:

Group Induction
1 (1-4) Vehicle control MEK (NOTOX Project 371216)
2 (5-8) Experimental Lowest test substance concentraton 1 %
3 (9-12) Experimental Intermediate test substance concentration: 10 %
4 (13-16) Experimental Highest test substance test concentration: 50 %
Extension
5 (17-20) Vehicle control MEK
6 (21-24) Experimental Lowest test substance concentraton 1 %

- Induction, Days 1, 2 and 3:
The dorsal surgace of both ears was epidermally treated (25 µL/ear( with each test substance concentration, approximately the same time each day. The 50 % concentrations was applied with a spatula.
- Treatment, Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 µCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intra-peritoneal injection with an overdose of pentobarbital. 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 were recorded. The nodes were pooled for each animal in 3 mL PBS.
- Tissue processing for radioactivity: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel guaze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. The DNA was precipitated with 3 mL 5 % trichloroacetic acid (TCA) at 4 °C for approximately 18 hours.
Precipitates were recovered by centrifugation at 200 g for 10 minutes, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold (Packard) as the scintillation fluid.
- Radioactivity measurements: All radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precisions of ± 0.2 % or a maximum of 5 minutes whichever came first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM/animal values were presented for each dose group and a Stimulation Index (SI) was calculated for each experimental group. The SI is the ration of the mean DPM/animal for each group against the mean DPM/animal of the vehicle group or the untreated group if the applied test substance concentration is 100%.
Where possible, an EC3 value (the estimated test substance concentration that gave SI = 3) was determined using linear interpolation.

Results and discussion

Positive control results:
Group Treatment Induction Mean DPM ± SD SI ± SD
1 Vehicle control Vehicle 149 ± 84 1
2 Positive control 5 % test substance 243 ± 79 2.0 ± 0.8
3 Positive control 10 % test substance 515 ± 362 4.2 ± 1.0
4 Positive control 25 % test substance 479 ± 425 3.9 ± 1.1

The size of the right node of one experimental animal treated with a 10 % HCA concentration was extremely large. The concurrent high DPM value obtained for this animal was considered to be an outlier and therefore rejected.
The SI values calcualted for the substance concentration 5, 10 and 25 % were 2.0, 4.2 and 3.9 respectively.
An EC3 value of 7.3 % was calculated using linear interpolation.
The stimulation index found at 25 % HCA was lower than expected, resulting in a poor dose response relationship. The calculated EC3 value of 7.3 % exceeded the minimum acceptable EC3 calue of 5% and was just below the range of EC3 values (7.7 - 8.8 %) found for HCA at the testing laboratory for the preceeding three years.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI of 3.1, 1.8 and 2.0 for 1, 10 and 50% concentrations respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM/animal values in test groups treated with HSY-2701 at 1, 10 or 50% were quite variable, and showed no clear dose-response relationship; SI values calculated from the test group means were 3.1, 1.8 and 2.0 respectively. However, the SI value of 3.1 for the 1% treated group was based on a mean which excluded one clearly defective radioactivity count (0.1 DPM for animal 7) but included another count that was unexpectedly high (279 DPM for animal 8). The results obtained after treatment with 1% HSY-2701 were therefore considered questionable. Accordingly, additional groups of animals were treated with vehicle and a 1% test substance concentration respectively. In this extended study, mice treated with HSY-2701 at 1% gave DPM/animal values greater than vehicle control values were considered to be implausible high, but even when the test group SI values was calculated using a control group mean excluding these two high values the resultant SI was only 0.6.

Any other information on results incl. tables

In the attached image, a dose reponse curve is presented as figure 1.

PRELIMINARY IRRITATION STUDY

Staining by the test substance prevented scoring for erythema. As no signs of necrosis were present, a 50% concentration was selected as highest concentration to be using in the main study.

Table 1: Radioactivity measurements (individual animals)

Group

Animal #

Treatment

Induction

DPM/animal

1

1

Vehicle control

MEK

17*

2

59*

3

73*

4

52*

2

5

Experimental

1% test substance

98

6

87

7

0.1#

8

279

3

9

Experimental

10% test substance

198

10

46

11

48

12

65

4

13

Experimental

50% test substance

107

14

186

15

45

16

62

5

17

Vehicle control

MEK

162

18

324

19

1530#

20

6157#

6

21

Experimental

1% test substance

243

22

141

23

105

24

116

* Data obstained from NOTOX project 37216

# Value not used for calculation of stimulation index

 

Table 2: Calculation of Stimulation Index (SI)

Group

Treatment

Induction

Mean DPM ± SD

SI ± SD

1

Vehicle control

MEK

50 ± 24

1

2

Experimental

1% test substance

155 ± 108*

3.1 ± 0.8

3

Experimental

10% test substance

89 ± 73

1.8 ± 0.9

4

Experimental

50% test substance

100 ± 63

2.0 ± 0.8

5

Vehicle control

Mek

243 ± 115

1

6

Experimental

1% test substance

153 ± 77

0.6 ± 0.3

* Mean value excludes animal 7. Mean value including animal 7 = 116, giving an SI value of 2.3

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The SI values calcualted for the substance concentrations 1, 10 and 50 % on the first occasion of testing were 3.1, 1.8 and 2.0. However, the 3.1 value at 1 % was based on exclusion of one erroneous (low) DPM count and inclusion of one unexpectedly high DPM count.
When a 1 % concentration of HSY-2701 was tested again, with fresh vehicle controls, an SI value (against variable control results) of 0.6 was obtained. Overall it was concluded that no sensitising capacity is expected fro HSY-2701.
Executive summary:

In a GLP compliant study performed in accordance with the OECD guideline 429, the sensitisation potential of HSY-2701 was determined using the local lymph node assay in CBA mice. HSY-2701 was concluded to have no sensitising capacity under the conditions of the test.