Registration Dossier

Administrative data

Description of key information

Skin sensitization: Local Lymph Node Assay, mice (CBA/CaOlaHsd), female (OECD guideline 429): not sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-18 - 2015-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP OECD 429 Guideline study without relevant deviations on the registered substance itself.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 36 - 65 % instead of 45 – 65% for several hours. This deviation to the study plan, however, does not affect the validity of the study.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 36 - 65 % instead of 45 – 65% for several hours. This deviation to the study plan, however, does not affect the validity of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test: 11 - 12 weeks, 2nd pre-test and main study: 8 - 9 weeks
- Weight at study initiation: 17.1 - 21.3 g
- Housing: As group in Makrolon Type II (pre-test) / III (main study) cages, with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): relative humidity approx. 45-65% (except for several hours, see deviations)
- Photoperiod (hrs dark / hrs light): 12 / 12, artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
dimethylformamide
Concentration:
2.5, 5, and 10% (w/w) in DMF
The test item was placed into an appropriate container on a tared balance and DMF was added.
The different test item concentrations were prepared serially.
The preparations were made freshly before each dosing occasion.
No. of animals per dose:
2 females for each pre-test
4 females (nulliparous and non-pregnant) per dose group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 50% solution in DMF Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing).
- Irritation/Lymph node proliferation response: 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. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
From day 2 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). Additionally, the animals showed signs of systemic toxicity, such as ruffled fur, reduced spontaneous activity, eyelid closure, as well as visible ear swelling. Moreover, a slight body weight loss was observed in both animals over the course of the study (animal 1: 1.5%, animal 2: 2.5%).
Therefore, a second pre-test was performed using test item concentrations of 5 and 10%. At the tested concentrations the animals did not show any signs of systemic toxicity. From day 3 to 5 the animal treated with 10% test item concentration showed an erythema of the ear skin (Score 1). The animal treated with 5% showed an erythema of the ear skin (Score 1) on day 4.
Thus, the test item in the main study was assayed at 2.5, 5, and 10% (w/w). 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.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³HTdR integration
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Topical application
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% (w/w) in DMF. 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).
Administration of ³H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.1 μCi of ³H-methyl thymidine (equivalent to 80.2 μCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.
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 and for the DPM values (group mean DPM ± standard deviation).
Positive control results:
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)
See "Any other information on results"
Parameter:
SI
Remarks on result:
other: 1.00 (0% test item) 1.74 (2.5% test item) 1.84 (5% test item) 2.16 (10% test item)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 3511 (0% test item) 6093 (2.5% test item) 6466 (5% test item) 7568 (10% test item)

Results

 

Calculation and Results of Individual Data

 

Table: Results with test item

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BG a)

number of lymph nodes

DPM per lymph node b)

S.I.

---

BG I

13

---

---

---

---

---

BG II

12

---

---

---

---

0

1

3511

3498.5

8

437.3

1.00

2.5

2

6093

6080.5

8

760.1

1.74

5

3

6466

6453.5

8

806.7

1.84

10

4

7568

7555.5

8

944.4

2.16

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.

 

Viability / Mortality

No deaths occurred during the study period.

 

Clinical Signs

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

 

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, 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)

 

Table: Results with positive control

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BG a)

number of lymph nodes

DPM per lymph node b)

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

 

Table: 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.

Interpretation of results:
GHS criteria not met
Conclusions:
The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitizing potential of 3-Methyl-thiazolidin-2-thion. Under the experimental conditions reported, the test item did not induce a stimulation index over 3 in test concentrations limited by irritating effects. Therefore, 3-Methyl-thiazolidin-2-thion is considered to not a skin sensitiser under the test conditions of this study.
Executive summary:

In a dermal sensitization study (OECD 429) with 3-Methyl-thiazolidin-2-thion in DMF, 8 - 9 weeks old female CBA/CaOlaHsd mice were tested in a local lymph node assay. α-Hexylcinnamaldehyde served as positive control. No deaths, no relevant body weights changes, no symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. In this study, 3-Methyl-thiazolidin-2-thion is not a dermal sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

There is a study available which was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin irritating potential of 3-Methyl-thiazolidin-2-thion. Under the experimental conditions reported, 3-Methyl-thiazolidin-2-thion is considered to not a skin sensitiser. There is no indicating given that the available result is not relevant for human risk assessment, the study is of high quality, no data gaps were identified. Hence, no additional testing is required and during risk assessment the substance can be regarded as not sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In the available local lymph node assay, Stimulation Indices of 1.74, 1.84, and 2.16 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in DMF. A dose response was 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. 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. As the determined S.I. is below 3, the substance does not need to be classified as skin sensitizer, and hence, classification as skin sensitizer is not needed.