Selenium disulphide

Endpoint summary

Description of key information

The substance is deemed to not be a sensitiser on the basis of a weight of evidence approach. Please refer to section 7.10.4 - "Sensitisation Data (humans) below for further information.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

Investigation of testing for the skin sensitization endpoint was undertaken initially using in vitro / in chemico techniques. The following was concluded:
The OECD 442D study guidelines quotes as follows:
The test method is applicable to test chemicals soluble or that form a stable dispersion (i.e. a colloid or suspension in which the test chemical does not settle or separate from the solvent into different phases) either in water or DMSO (including all of the test chemical components in the case of testing a multi-constituent substance or a mixture). Test chemicals that do not fulfil these conditions at the highest final required concentration of 2000 μM may still be tested at lower concentrations. In such a case, results fulfilling the criteria for positivity could still be used to support the identification of the test chemical as a skin sensitiser, whereas a negative result obtained with concentrations < 1000 μM should be considered as inconclusive.
As the test substance could not be dissolved in the test media, this test could not be conducted..
Furthermore, consideration of the OECD 442E study states as follows:
The h-CLAT method is applicable to test chemicals soluble or that form a stable dispersion (i.e. a colloid or suspension in which the test chemical does not settle or separate from the solvent/vehicle into different phases) in an appropriate solvent/vehicle. .
As the test substance could not be dissolved in the test media, this test could not be conducted..
Due consideration was therefore given to how to assess the sensitization endpoint.. Conducting the OECD 442C study was considered to be of little value if the additional two required tests could not be conducted. As the requirements for sensitization in REACH are the above 3 in Annex VII 8.3.1 are as follows:
— the available in vitro/in chemico test methods are not applicable for the substance or are not adequate for classification and risk assessment according to point 8.3.
An in vivo study shall be conducted only if in vitro/in chemico test methods described under point 8.3.1 are not applicable, or the results obtained from those studies are not adequate for classification and risk assessment according to point 8.3.
It was considered that the in vitro studies were not suitable for classification and risk assessment of this substance, and the decision to proceed with a LLNA test was considered appropriate.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

17 January 2018 - 27 March 2018.

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was conducted in accordance to relevant test guidelines in a GLP accredited laboratory. However, as there was a poor dose response an EC3 value could not be calculated but was instead estimated. The results are considered to be indicative of a “false positive” as they do not follow the known trend for the selenium containing group of substances and is therefore discarded. Please refer to Section 13.2 - Weight of Evidence Approach for the Assessment of Skin Sensitisation Potential of Selenium Disulphide, CAS 7488-56-4, EC 231-303-8 for further information.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables"

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further specific details.

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of relevant OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals/groups
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 18.1-19.6 grams (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time:at least 7 days
Note: In the preliminary experiments mice of 9-10 weeks of age (17.4-18.4 g) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the staff Veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 15.3 – 28.4 °C
Relative humidity: 23 - 96 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 382 24962, Expiry date: 30 April 2018, respectively; produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch numbers: 60172230020101 and 60180640040101, Expiry dates: 11 August 2018 and 05 March 2019, respectively) produced by Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water taken from the municipal supply and provided in a 500 ml bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u.36, Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Citoxlab Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study. Certified Nest building material was also provided for animals (Arbocel produced by J. Rettenmaier & Söhne GmbH + Co.KG).

Identification
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software according to the actual body weights, verifying the homogeneity and variability between the groups.

Vehicle:

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

Concentration:

Preliminary study test concentrations: 25, 10, 5, 2.5 and 1 %
Main study test concentrations: 2.5, 1, 0.5 and 0.25 %

No. of animals per dose:

4 animals per dose

Details on study design:

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Tests were conducted according to the Study Plan on CBA/CaOlaHsd mice using five doses (2 animals/dose) at test item concentrations of 25, 10, 5, 2.5 and 1 % (w/v) in AOO. The preliminary experiments were conducted in a similar experimental manner to the main study, but were terminated on Day 6 and the radioactive proliferation assay was not performed.

The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum achievable concentration was 25 % (w/v).

In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored according to OECD Guidelines for Testing of Chemicals No. 404, Table 2 [3]. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (before treatment, approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

During the Preliminary Irritation / Toxicity Tests, signs of systemic toxicity (slight to severe decrease of activity (5 out of 5 animals) and hunched back (4 out of 5 animals)) followed by mortality was observed in the 25, 10 % (w/v) (2 out of 2 animals, respectively), and in the 5 % (w/v) dose group (1 out of 2 animals). All surviving animals were symptom free. Clinical observations are summarized in Table 8 of Appendix 3 and Table 11 of Appendix 4.

No marked body weight loss (>5% reduction of body weight) was observed in any of the surviving animals at Day 6 (3 days after the last treatment) in the preliminary study (Table 6 of Appendix 3 and Table 9 of Appendix 4).

Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear thickness values and the weights of the ear punches (2 per animal) are summarized in Table 7 of Appendix 3 and Table 10 of Appendix 4.

The ear thickness values on Day 6 were more than 25 % higher compared to Day 1 for the one surviving animal at the 5 % (w/v) dose group, and for both animals at the 2.5 % (w/v) dose group. Ear punch weights in the surviving animals were elevated, but were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in the surviving 5 % (w/v) animal and in the 2.5 % (w/v) dose group; and normal in the 1 % (w/v) dose group (subjective judgement by analogy with observations of former experiments).

Based on these results, 2.5 % (w/v) dose is selected as top dose for the main test.

Topical application
During the assay, each mouse was topically dosed on the dorsal surface of each ear with 25 µL of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). Formulations were stirred from preparation up until the treatment such that samples taken for dosing were homogenous There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatment) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (±30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).

The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.

Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 C.

After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, samples were centrifuged and supernatants were removed. Pellets were gently resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.

After overnight (approximately 18 hours) incubation at 2-8 °C, precipitations were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).

The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

USE OF RADIOACTIVE MATERIALS
Use of radioactive materials was recorded in the appropriate register. Regular decontamination of the working area with a verification of decontamination was carried out. Radioactive waste materials were processed according to normal laboratory standards.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (stimulation index value of 8.6) was noted for a-Hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.

The observed mean DPN values for the negative and positive control were within the historical control range. Historical control data for the positive and negative control substances

Key result

Parameter:

EC3

Remarks on result:

not determinable because of methodological limitations

Cellular proliferation data / Observations:

Appearance of the lymph nodes were larger than normal in the 2.5, 1 % (w/v) dose groups, in 2 out of 4 animals in the 0.5 % (w/v) dose group, and in the positive control group. Slightly larger than normal lymph nodes were observed in 2 out of 4 animals of the 0.5 % group and in all animals of the 0.25 % (w/v) dose group. Normal lymph nodes were observed in the negative control group.

The data show the test item is conclusively positive in this assay, however the poor dose response at the lower concentrations prevents an estimate of the EC3 being extrapolated.

The EC3 could not be calculated from the data but was clearly below 2.5% (w/v ).

CLINICAL OBSERVATION

No mortality or systemic toxicity was observed during the main study. Test item precipitate or minimal test item precipitate was present on the ears of the animals in the 2.5, and 1.0 % (w/v) groups (4 out of 4 animals, respectively).

BODY WEIGHT MEASUREMENT       

No treatment related effects were observed on the mean body weight of the main study groups.

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change^# (%)
5768	1	Negative (vehicle) control(in AOO)	19.2	19.8	3.1
5772	2		18.7	18.3	-2.1
5781	3		18.4	19.2	4.3
5784	4		18.8	19.1	1.6
		Mean	18.8	19.1	1.7
5773	5	Selenium Disulphide 2.5 (w/v) % (in AOO)	19.6	20.2	3.1
5766	6		19.4	19.0	-2.1
5782	7		18.3	17.9	-2.2
5774	8		18.3	19.5	6.6
		Mean	18.9	19.2	1.3
5780	9	Selenium Disulphide 1 (w/v) % (in AOO)	19.5	20.0	2.6
5767	10		18.1	19.0	5.0
5779	11		18.8	19.1	1.6
5788	12		18.5	19.1	3.2
		Mean	18.7	19.3	3.1
5778	13	Selenium Disulphide 0.5 (w/v) % (in AOO)	19.5	19.3	-1.0
5770	14		18.9	19.5	3.2
5783	15		18.2	18.6	2.2
5771	16		18.4	18.5	0.5
		Mean	18.8	19.0	1.2
5777	17	Selenium Disulphide 0.25 (w/v) % (in AOO)	19.2	19.4	1.0
5769	18		19.3	19.5	1.0
5785	19		18.7	20.0	7.0
5789	20		18.7	19.3	3.2
		Mean	19.0	19.6	3.1
5776	21	Positive control (25% (w/v) HCA in AOO)	19.6	20.0	2.0
5786	22		18.7	18.9	1.1
5787	23		18.1	18.9	4.4
5775	24		18.2	18.8	3.3
		Mean	18.7	19.2	2.7

Notes:

1. *: Terminal body weights were measured on Day 6.

2. #: = (Terminal Body Weight – Initial Body Weight) x 100 / Initial Body Weight

DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM/group	DPM	No. of Nodes	DPN	Stimulation Index Values
Background (5 (w/v) % TCA)	41	-	-	-	-
Negative control (AOO)	3392	3351.0	8	418.9	1.0
Selenium Disulphide 2.5 % (w/v) in AOO	22113	22072.0	8	2759.0	6.6
Selenium Disulphide 1 % (w/v) in AOO	21545	21504.0	8	2688.0	6.4
Selenium Disulphide 0.5 % (w/v) in AOO	15103	15062.0	8	1882.8	4.5
Selenium Disulphide 0.25 % (w/v) in AOO	18376	18335.0	8	2291.9	5.5
Positive control (25 % HCA in AOO)	28972	28931.0	8	3616.4	8.6

INTERPRETATION OF OBSERVATIONS

The test item was a powder, which was formulated in AOO. There were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions were considered to be evidence that Selenium Disulphide caused lymphoproliferation, and the size of lymph nodes were in good correlation with this conclusion.

The data show the test item is conclusively positive in this assay, however the poor dose response at the lower concentrations prevents an estimate of the EC3 being extrapolated.

The EC3 could not be calculated from the data but was clearly below 2.5% (w/v ).

Interpretation of results:

study cannot be used for classification

Conclusions:

The weight of evidence approach to the assessment of selenium disulphide utilising human monitoring data, selenium read-across substances and literature review; SeS2 cannot be regarded as a skin senstiserbased on the LLNA study conducted. The insolubility of the SeS2 does not allow for accurate and reliable LLNA studies to be performed as it gives rises to non-homogenous suspensions, in as such the LLNA study which was conducted is predicted to be a false positive.

Executive summary:

The weight of evidence approach to the assessment of selenium disulphide utilising human monitoring data, selenium read-across substances and literature review; SeS2 cannot be regarded as a skin sensitiser based on the LLNA study conducted. The insolubility of the SeS2 does not allow for accurate and reliable LLNA studies to be performed as it gives rises to non-homogenous suspensions, in as such the LLNA study which was conducted is predicted to be a false positive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The weight of evidence approach to the assessment of selenium disulphide utilising human monitoring data, selenium read-across substances and literature review; SeS₂cannot be regarded as a skin sensitiser based on the LLNA study conducted. The insolubility of the SeS₂does not allow for accurate and reliable LLNA studies to be performed as it gives rises to non-homogenous suspensions, in as such the LLNA study which was conducted is predicted to be a false positive. Peer reviewed human data indicates that sensitisation is not anticipated at the levels of use of the substance in formulated products. The substance is therefore considered not to demonstrate sensitising properties and this hazard is not applicable to the substance in question. Please refer to Section 7.10.4 Sensitisation data (humans) below.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available