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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:
24 June-16 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with international guidelines

Data source

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

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
Test material form:
other: pastilles
Details on test material:
Identification: SABOSTAT A 300
Batch: 20085596
Purity: 100%
Appearance: Whitish pastilles
Expiry Date: 10 April 2016
Storage Conditions:
(provided by the Sponsor) At room temperature, moisture protected, light protected
Stability in Solvent: Not indicated by the sponsor
Purpose of Use: Chemical

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Harlan Laboratories B.V.
Postbus 6174
5960 AD Horst / The Netherlands
Number of animals for
pre-test: 4 females
Number of animals for
the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): 1st and 2nd pre-test: 9 - 10 weeks
Main study: 8 - 9 weeks
Body weight: see Appendix 1 and 2
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet
(certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
(except for several hours, see deviations)
relative humidity approx. 45-65%
(except for several hours, see deviations)
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
other: cotton seed oil
Concentration:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50% solution in cotton seed oil. Warming to approximately 37°C was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days.
At the tested concentrations the animals did not show any signs of systemic toxicity or body weight loss. However both animals showed signs of excessive local skin irritation (erythema score 3, increase in ear swelling ≥25%), for details see Appendix 1.
Therefore, a second pre-test was performed using test item concentrations of 5 and 10%. At these concentrations, the animals showed a very slight to well defined erythema of the ear skin (score: 1-2, for details see Appendix 1), as well as a slight erythema of the scalp. Furthermore, increased spontaneous activity was observed in both animals from day 3 onwards, which was attributed to the irritative properties of the test item. However no excessive local skin irritation as defined by OECD 429 was present in both animals
Thus, the test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

No. of animals per dose:
4
Details on study design:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in cotton seed oil. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.7 µCi of 3H-methyl thymidine (equivalent to 78.7 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables

Results and discussion

Positive control results:
see field "Any other information on results including tables"

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
4
Remarks on result:
other: see Remark
Remarks:
test item concentration: 0%
Parameter:
SI
Value:
1.36
Test group / Remarks:
4
Remarks on result:
other: see Remark
Remarks:
test item concentration 2.5%
Parameter:
SI
Value:
0.93
Test group / Remarks:
4
Remarks on result:
other:
Remarks:
test item concentration: 5%
Parameter:
SI
Value:
20.8
Test group / Remarks:
4
Remarks on result:
other:
Remarks:
test item concentration: 10%

Any other information on results incl. tables

Results

Calculation and Results of Individual Data

Vehicle: cotton seed oil

 

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

21

---

---

---

---

---

BG II

26

---

---

---

---

0

1

25830

25806.5

8

3225.8

1.00

2.5

2

35079

35055.5

8

4381.9

1.36

5

3

24033

24009.5

8

3001.2

0.93

10

4

53752

53728.5

8

6716.1

2.08

1    =  Control Group

2-4=  Test Group

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

No deaths occurred during the study period.

No signs of systemic toxicity were observed during the study period.On days 1 to 4, the animals treated witha test item concentration of10% showed an erythema of the ear skin (Score 1). The animals treated with a test item concentration of 5% showed an erythema of the ear skin on day 3 only. An erythema of the scalp (score: 1) was noted in the animals treated with 5 and 10%, respectively, on day 3 only. Animals treated with 2.5% test item concentration did not show any signs of local skin irritation.

The body weight of the animals, recordedprior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

Results of the GLP Positive Control

Experiment performed in April 2015 (Harlan study number 1690900). Positive control substance: α-Hexylcinnamaldehyde

Vehicle: acetone:olive oil (4+1, v/v)

 

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

15

---

---

---

---

---

BG II

11

---

---

---

---

0

1

6228

6215.0

8

776.9

1.00

5

2

12016

12003.0

8

1500.4

1.93

10

3

16484

16471.0

8

2058.9

2.65

25

4

58951

58938.0

8

7367.3

9.48

1    =  Control Group

2-4=  Test Group

a)   =  The value was taken from the figure BG

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Calculation of the EC3 value:

 

Test item concentration %

S.I.

Test Group 3

10 (a)

2.65 (b)

Test Group 4

25 (c)

9.48 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 10.8% (w/v)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.36, 0.93 and 2.08 were determined with the test item at concentrations of 2.5, 5, and 10% in cotton seed oil. A dose response was not observed.
The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.
The substance was not a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item formulated in cotton seed oil was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On days 1 to 4, the animals treated witha test item concentration of10% showed an erythema of the ear skin (Score 1). The animals treated with a test item concentration of 5% showed an erythema of the ear skin on day 3 only. Furthermore, erythema of the scalp was noted in the animal treated with 5 and 10%, respectively. Animals treated with 2.5% test item concentration did not show any signs of local skin irritation.

In this study Stimulation Indices (S.I.) of 1.36, 0.93 and 2.08 were determined with the test item at concentrations of 2.5, 5, and 10% in cotton seed oil, respectively.

The substance was not a skin sensitiser under the test conditions of this study.