Dimethyl disulphide

Endpoint summary

Description of key information

The skin sensitisation potential of DMDS was evaluated in two LLNA assays. In the assay with the technical DMDS (purity 99.8%), DMDS induced a positive response but a clear EC3 value was not determined. In the assay with a plant protection formulation containing 93.1% w/w of DMDS, the EC3 value was 22.41% indicating a weak sensitiser potential further supported by 2 negative sensitisation tests by repeated dermal application.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 February 2012 - 04 April 2012

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)

Version / remarks:

2010

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
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the beginning of the treatment period, the animals of the preliminary test were approximately 10-12 weeks old and the animals of the main test were approximately 8 weeks old
- Mean body weight at study initiation: in the main test, they had a mean body weight of 20.0 g (range: 18.8 g to 21.0 g)
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 6 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: 15 February 2012 to 27 February 2012.

Vehicle:

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

Concentration:

Range-finding test: 10%, 25%, 50% and 100%.
Main test: 2.5%, 5%, 10%, 25% and 50%.

No. of animals per dose:

4 per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: In order to select the most appropriate concentration, the solubility assay first started at the concentration of 50%.
A solution was obtained at the concentration of 50% in AOO.
As the test item is a liquid that can be sampled using a pipette, the maximum achievable concentration was 100%.
- Irritation: Female treated at 100% was found dead on day 3.
Dryness of the skin was noted on day 2 in female treated at 100%.
No notable increase in ear thickness was observed at any tested concentrations.
The highest concentration retained for the main test was therefore 50%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Positive control substance(s):

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

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

Parameter:

EC3

Value:

ca. 2.5

Parameter:

SI

Value:

ca. 2.99 - ca. 3.38

Test group / Remarks:

2.5%

Parameter:

SI

Value:

ca. 2.4 - ca. 2.4

Test group / Remarks:

5%

Parameter:

SI

Value:

ca. 1.85 - ca. 1.9

Test group / Remarks:

10%

Parameter:

SI

Value:

ca. 3.3 - ca. 3.58

Test group / Remarks:

25%

Parameter:

SI

Value:

ca. 4.75 - ca. 4.77

Test group / Remarks:

50%

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	2.5	I	2.99 / 3.38
Test item	5	I	2.40 / 2.40
Test item	10	I	1.90 / 1.85
Test item	25	I	3.30 / 3.58
Test item	50	I	4.75 / 4.77
HCA	25	-	20.98 / 19.98

-: not recorded

I: non-irritant (increase in ear thickness < 10%)

…/…: first counting / second counting

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Dimethyl disulphide induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained, the test item should be considered as a moderate sensitizer.

Executive summary:

The objective of this study was to evaluate the potential of dimethyl disulphide to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with the principles of Good Laboratory Practice.

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Four groups of one female mouse received the test item by topical route to the dorsal surface of both ears (one concentration per animal) on days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thicknessof both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. On completion of the observation period, surviving animals were sacrificed then discarded without macroscopic post-mortem examination.

In the main test, five groups of four female mice received the test item by topical routeto the dorsal surface of both earson days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (acetone/olive oil (4/1; v/v)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.

From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and then on days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (³H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of³H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

No unscheduled deaths and no clinical signs were observed during the observation period.  

No local reactions were observed in any animals.

No notable increase in ear thickness was observed at any tested concentrations.

The SI of the positive control was > 3; this experiment was therefore considered valid.

A significant lymphoproliferation was noted at the concentration of 2.5% (SI = 3) and then at the concentrations of 25% and 50% (SI > 3).

In view of the absence of dose-response relationship, it was decided to perform a second counting in order to check the dmp values.

The first results were confirmed. A significant lymphoproliferation (SI > 3) was noted at the concentrations of 2.5, 25 and 50%.

Therefore, despite the absence of dose-response relationship and as no local irritation was observed, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

The EC₃value is approximately 2.50%.

Dimethyl disulphide induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC₃value obtained, the test item should be considered as a moderate sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Version / remarks:

2010

Deviations:

yes

Remarks:

the animals were individually identified on the tail using an indelible marker instead of being identified by an implanted microchip.

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

idem above

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
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 8 weeks old on the day of treatment
- Mean body weight at study initiation: 19.8 g (range: 18.5 g to 21.1 g)
- Fasting period before study: no
- Housing: the animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the animals were acclimated to the study conditions 13 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: 13 October 2011 to 08 November 2011

Vehicle:

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

Concentration:

2.5, 5, 10, 25 and 50%.

No. of animals per dose:

4 per dose.

Details on study design:

RANGE FINDING TESTS:
A solution was obtained at the concentration of 50% in AOO.
As the test item is a liquid that can be sampled using a pipette, the maximum achievable concentration was 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness.

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Positive control substance(s):

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

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

Parameter:

EC3

Value:

22.42

Parameter:

SI

Value:

0.91

Test group / Remarks:

2.5%

Parameter:

SI

Value:

1.07

Test group / Remarks:

5%

Parameter:

SI

Value:

0.79

Test group / Remarks:

10%

Parameter:

SI

Value:

3.46

Test group / Remarks:

25%

Parameter:

SI

Value:

4.1

Test group / Remarks:

50%

Parameter:

SI

Value:

4.83

Test group / Remarks:

Positive control

Cellular proliferation data / Observations:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 4.83). The experiment was therefore considered as valid.
A significant lymphoproliferation (SI > 3) was noted at the concentrations of 25 and 50% with a
dose-relationship.
In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.
The EC3 value is equal to 22.42%.


CELLULAR PROLIFERATION DATA
DPM per group: Group 1: Vehicle: 2729 Group 2: 2.5%: 2475 Group 3: 5%: 2925 Group 4: 10%: 2153 Group 5: 25%: 9445 Group 6: 50%: 11192

CLINICAL OBSERVATIONS:
No clinical signs were observed in any animals.
No local reactions were observed in any animals.
No notable increase in ear thickness was observed at any tested concentration.

BODY WEIGHTS
The body weight change of test item-treated animals was similar to that of control animals.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Atomal13 induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained, the test item should be considered as a weak sensitizer.

Executive summary:

The potential of Atomal13 to induce delayed contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the OECD guideline no. 429 and the principles of Good Laboratory Practice.

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, 28 female CBA/J mice were allocated to 7 groups:

.            five treated groups of four animals receiving the test item at the concentration of 2.5, 5, 10, 25 or 50% in Acetone/Olive Oil (AOO)(vehicle),

.            one negative control group of four animals receiving the vehicle,

.            one positive control group of four animals receiving the positive control item,a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v).

During the induction phase, the test item, vehicle or positive control item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

In the preliminary test, one female from group 2 given test item at concentrations of 50 and 100% was found dead on day 2. No clinical signs were observed during the preliminary test. No unscheduled deaths or clinical signs were observed during the main test. No cutaneous reactions or increase in ear thickness were observed at any of the tested concentrations.

The acceptance criterion was met; this experiment was therefore considered as valid. The results are presented in the following table:

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	2.5	non-irritant	0.91
Test item	5	non-irritant	1.07
Test item	10	non-irritant	0.79
Test item	25	non-irritant	3.46
Test item	50	non-irritant	4.10
HCA	25	-	4.83

A significant lymphoproliferation (SI > 3) was noted at the concentrations of 25% and 50% with a dose-relationship.

In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

The EC₃value is equal to 22.42%.

Atomal13 induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC₃value obtained, the test item should be considered as a weak sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo studies
An OECD test guideline no. 429 compliant GLP study was conducted to investigate the potential of Dimethyl disulphide (DMDS, purity 99.8%) to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA) (Rokh, 2012). On the basis of a preliminary study, 2.5, 5, 10, 25 and 50% DMDS concentrations were selected and the vehicle was 4/1 acetone/olive oil. A positive control group (a-hexylcinnamaldehyde (HCA)) was included in the study for validation purposes. During the induction phase, DMDS, vehicle or HCA was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3) and after 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.
 In the preliminary test one animal treated topically with 100% DMDS was found dead on day 3. The cause of death was not established. In the main test DMDS had no effect on body weight, was not associated with clinical signs or local reactions and did not increased ear thickness.
The acceptance criteria for the positive control (Stimulation index =3) was met, thus the study was considered valid. In view of the absence of a dose-response relationship a second counting was performed to check the disintegration count results; the values obtained at the first count were confirmed. A significant lymphoproliferation (SI > 3) was noted at DMDS concentrations of 2.5, 25 and 50%. Therefore, despite the absence of a dose-response relationship and in the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value is approximately 2.5%.

It may therefore be concluded that under the experimental conditions of this study, DMDS (purity 99.8%) induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained (approximately 2.5%), the test item should be considered as a moderate sensitiser.

 

The potential of Atomal13 (plant protection formulation containing 93.1% w/w of DMDS) to induce delayed contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA) (Watzinger, 2011). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice. A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, 28 female CBA/J mice were allocated to 7 groups: five treated groups of four animals receiving the test item at the concentration of 2.5, 5, 10, 25 or 50% in Acetone/Olive Oil (AOO)(vehicle), one negative control group of four animals receiving the vehicle, one positive control group of four animals receiving the positive control item,a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v). During the induction phase, the test item, vehicle or positive control item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

In the preliminary test, one female from group 2 given test item at concentrations of 50 and 100% was found dead on day 2. No clinical signs were observed during the preliminary test. No unscheduled deaths or clinical signs were observed during the main test. No cutaneous reactions or increase in ear thickness were observed at any of the tested concentrations. The acceptance criterion was met; this experiment was therefore considered as valid.

A significant lymphoproliferation (SI > 3) was noted at the concentrations of 25% and 50% with a dose-relationship. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value is equal to 22.42%. The test item Atomal13 induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained, the test item should be considered as a weak sensitizer.

 

The allergy sensitization potential of Dimethyl Disulfide was evaluated following repeated dermal exposure (Hall, 2015). This study was designed to comply with the standards set forth in the National Standard of the People's Republic of China Toxicological Test Methods of Pesticides for Registration (GB 15670-1995). This study was conducted in accordance with the Good Laboratory Practices regulations of the USEPA. Fifteen healthy male and fifteen healthy female Hartley Albino guinea pigs were assigned to the study. Group 1 (10 males and 10 females) was induced with Dimethyl disulfide at a concentration of 100%. Group 2 (5 males and 5 females) was not dosed and served as a naive control. Based on the results of Group 1, 100% was chosen as the highest non-irritating concentration for the challenge Phase and was administered to both groups two weeks after the third induction. During the induction phase, group 1 received three dose applications, 0.1 ml per site, one per week for three weeks. The test sites (#1) were scored for dermal irritation at 24 and 48 hours after patch removal. During the challenge phases, groups 1 and 2 received one dose application, 0.1 ml per site. The test sites (#2) were scored for dermal irritation daily, beginning at 24 hours after patch removal, for 12 consecutive days. Since there was no reaction at the initial challenge sites, Groups 1 and 2 were dosed again in the same. The test sites (#3) were scored for dermal irritation at 24, 48 and 72 hours after patch removal. Body weights were recorded pretest and at termination. All animals were observed once per day for mortality, toxicity and systemic observations. The sensitivity of guinea pigs to a positive control, 85% a-Hexylcinnamaldehyde (HCA) is confirmed in this laboratory (approximately every six months).

During inductions 1 to 3, erythema was absent. Days 1 to 12 after the 1st challenge, erythema was absent in both the induced and naive control groups. At 24, 48 and 72 hours after the 2nd challenge, erythema was absent in both the induced and naive control groups. Chromorhinorrhea, soiling, wetness and yellow staining of the anogenital area were observed in both the test article and naive control groups. All animals gained body weight.

The Sensitization Strength Classification of Dimethyl disulfide is weak allergens (Grade 1).

 

The delayed contact hypersensivity of dimethyl disulphide was evaluated in Guinea pigs according to EPA-40 CFR 163-81-6 guideline (Buehler test) (Shapiro, 1985). The induction phase with undiluted dimethyl disulphide has been realized by cutaneous route every alternate day during 10 days in a group of 10 guinea pigs. The challenge phase was realized 14th days after the last application by cutaneous application of undiluted dimethyl disulphide; the cutaneous reactions were scored 24 and 48 hours after the challenge phase. No skin reactions related to a contact sensitiziation was observed. Dimethyl disulphide was not sensitizing in guinea pigs.

In vitro studies
A combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of DMDS.
• protein reactivity (DPRA),
• activation of keratinocytes (LuSens), and
• activation of dendritic cells (MUSST).
The results of the individual studies are summarized and evaluated to predict the presence or absence of skin sensitizing potential of DMDS. The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al. , 2012. Based on the performance standards of the OECD test guideline no. 429 (Local Lymph Node Assay, LLNA) , the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%). A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the present time. Based on the results summarized and applying the evaluation criteria, DMDS is predicted to be a skin sensitizer.

- in vitro DPRA
The reactivity of DMDS towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA) (BASF, 2013a). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.
The following results were obtained in the DPRA:
The test substance was solved in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.
The mean C-peptide depletion, caused by the test substance was determined to be 96.80%. The mean K-peptide depletion, caused by the test substance was determined to be 0.76%. Thus, the mean peptide depletion was calculated to be 48.78%. No co-elution of test substance and peptides was noticed.
Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) and cited in chapter 3.10 it was concluded that DMDS shows a high chemical reactivity in the DPRA under the test conditions chosen.

- in vitro MUSST
The potential of test substance DMDS to induce the cell membrane marker CD86 expression was evaluated in the Myeloid U937 Skin Sensitization Test (MUSST) (BASF, 2013b). For this purpose the test substance was incubated with human pro-monocytic cell line U937 for ca. 48 hours at 37°C and membrane marker expression was measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5 µg/mL up to 2000 µg/mL) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test after 48 hour exposure U937 cells were stained with FITC labeled antihuman-CD86 antibody and propidium iodide, the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid experiments were performed.
The test substance was soluble in DMSO (400 x stock solutions) and soluble in 0.25% DMSO (final concentrations).
After 48 hours precipitates were not noticed in any concentration. After 48 hours of exposure to test substance DMDS, CD86 expression was induced in U937 cells affording at least 70% viability in two independent experiments. From this it has to be concluded that test substance DMDS does induce dendritic cell activation.

- ARE Reporter Assay - LuSens
The keratinocyte activating potential of test substance DMDS was evaluated in the LuSens assay (BASF, 2013c). For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined thereafter by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The following results were observed:
The test substance (100 x stock solutions) was soluble in DMSO and soluble in 1% DMSO (final concentrations). After 48 hours precipitates were not noticed in any concentration. After 48 hours of exposure to DMDS luciferase activity in LuSens cells was not induced affording at least 70% viability in at least two independent experiments. From this it has to be concluded that DMDS does not have a keratinocyte activating potential.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available date, DMSO is classified as skin sens.1 (H317 : May cause allergic reactions) according to the Regulation EC n°1272/2008. The determination of the sub-category is not possible according to the RAC.

Justification of classification (RAC) :

In the LLNA of Rokh (2012), performed with DMDS (purity 99.8%) no significant stimulation of proliferation of cells was observed at concentration of the 5% – 10%, but at a concentration of 2.5% an SI of approx. 3 was noted in two countings. It is uncertain whether this SI at a concentration 2.5% was in fact induced by DMDS since it was not observed at the two higher concentrations 5 and 10%. However, no concentration of ≤ 2 % was tested and based on the result of this study, classification in category 1A cannot be excluded.
In the LLNA performed with a plant protection product (named Atomal13) containing 93.1% of DMDS (Watzinger, 2011), no significant stimulation of proliferation of cells was observed at 2.5% – 10% concentration of the formulation. The concentrations of DMDS in these trials were even slightly lower noting that its content in the formulation was 93.1%. However, it is noted that Atomal13 is not a substance, but a mixture, and therefore even though this study provides evidence that EC3 for this plant protection product is above 10%, the category 1A for DMDS cannot be excluded.
In addition to the LLNA, the results of three in chemico /in vitro tests and the Buehler assay were used in weight of evidence analysis to assess skin sensitising potential of DMDS.
The results of DPRA (BASF, 2013a) evaluating the peptide reactivity of DMDS, which is the first key event of the skin sensitisation AOP, demonstrated high reactivity of DMDS in this test system. At present, there are no established rules for assessment of skin sensitising potency based on results of the positive test, so the results are taken as supportive evidence for skin sensitising potential of DMDS.
The antioxidant response element (ARE) was not induced in the LuSens assay (BASF, 2013c). This assay evaluates a potential of the test substance to induce cyto-protective gene pathways in keratinocytes (the second event in the skin sensitisation AOP). The result of the study suggests that this pathway was not or was only slightly activated by DMDS which together with negative results in the Buehler assay indicates that skin sensitising potential of DMDS is not very high.
Induction of specific cell surface marker CD86 in the U937 cells in the Myeloid U937 Skin Sensitisation Test (MUSST) (BASF, 2013b) indicates that DMDS activates the dendritic cells, which is the third key event of the skin sensitisation AOP. The results of this in vitro assay are taken as supportive evidence for skin sensitising potential of DMDS.