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Description of key information

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:
2010-04-27 till 2010-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: Mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.3 - 23.3 g
- Housing:single caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst / Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf), ad libitum
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)
- 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 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The highest test item concentration which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v).
In the pre-test, two mice were treated with test item concentrations of 50 or 100%.
The test item in the main study was assayed at 25, 50, and 100%.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v).
In the pre-test, two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 or 100% once daily each on three consecutive days. At those concentrations the animals did not show signs of systemic toxicity or local irritation.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4:1). The application volume, 25 µl, was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF ³H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED ³H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after treatment with ³HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. 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.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: Once daily (week day) from experimental start to necropsy.
- Body weights: In the pre-test prior to the first application and prior to sacrifice; in the main experiment prior to the first application and prior to treatment with 3HTdR.
- Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.
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.
Positive control results:
Experiment performend in November 2009:
5, 10, and 25% alpha-hexyl cinnamic aldehyde in acetone:olibe oil (4:1) yielded a S.I. of 1.78, 2.54, and 4.88, respectively. The EC3 value calculated was 12.9%.
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.
Parameter:
SI
Value:
1.22
Test group / Remarks:
25% - pooled samples
Remarks on result:
other: see Remark below Table
Remarks:
.
Parameter:
SI
Value:
0.99
Test group / Remarks:
pooled samples - 50%
Remarks on result:
other: see remark below Table
Parameter:
SI
Value:
3.46
Test group / Remarks:
pooled samples - 100%
Remarks on result:
other: see remark below Table
Cellular proliferation data / Observations:
Since the lymph nodes were pooled there is no information on individual lymph node results, outlier(s) may have been present.
Also, at the 100% concentration (pure chemical), no vehicle was used which may have influenced the outcome.

Results

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

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

17

---

---

---

---

---

BG II

17

---

---

---

---

---

1

3517

3500

8

437.5

---

25

2

4282

4265

8

533.1

1.22

50

3

3479

3462

8

432.8

0.99

100

4

12129

12112

8

1514.0

3.46

BG = background ( 1 mL 5% trichloroacetic acid) in duplicate

1 = control group

2 -4 = test groups

SI = Stimulation Index

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

b) since the lymph nodes of the animals of groups 1 -4 were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings 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.

 

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
At 100% (pure chemical) the SI was slightly above 3. However, as the lymph nodes were pooled there is no information on the presence of outliers. Moreover, pure chemical and no vehicle was used which may also have influenced the outcome. In the opinion of the applicant this is a false positive result and CS2 cannot be considered as a skin sensitizer based on this study.
Executive summary:

In this study carbon disulfide dissolved in acetone/olive oil (4:1 v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay according to OECD guideline 429 was performed using test item concentrations of 25, 50, and 100%. All treated animals survived the scheduled study period and no signs of systemic toxicity or local irritation were observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration results 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 1.22, 0.99, and 3.46 were determined with the test item at concentrations of 25, 50, and 100% in acetone/olive oil (4:1). Based on the outcome at 100%, carbon disulfide could be considered a skin sensitizer under the test conditions of this study. However, as the lymph nodes were pooled there is no information on the presence of outliers. Moreover, pure chemical and thus no vehicle was used which may also have influenced the outcome. In the opinion of the applicant this is a false positive result and CS2 cannot be considered as a skin sensitizer based on this study.

 

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

A Local Lymph Node Assay was performed, according to OECD Guidline 429. Female mice were tested at concentrations of 25, 50, and 100% (w/v); vehicle used acetone: olive oil (4:1). The concentrations were chosen based on a pre-test performed. Simulation indices (S.I.) relative to the mean control values were as follows: 1.22, 0.99, and 3.46. According to the guidance, when S.I. ≥ 3 in the LLNA test, the substance may be regarded as a skin sensitizer. However, the findings give a clearly abnormal dose-response and a weakly positive result was observed only at the highest concentration applied. However, because lymph nodes were pooled there is no information on the presence of outliers; in addition at 100% pure chemical and no vehicle was used which may have influenced the outcome. In the opinion of the applicant this is a false positive result. No other information was found, regarding the skin sensitising properties of CS2 in experimental animals.

A thorough assessment of the available human data shows that over a century of well-documented occupational skin exposure to CS2, it has not resulted in one single medical report of skin sensitisation.

Taken all together, CS2 shall not be considered a skin sensitiser.

 

Justification for classification or non-classification

Based on the discussion above, carbon disulfide does not need to be classified as a skin sensitiser.