Thionyl dichloride

Endpoint summary

Description of key information

Oral: LD50 = 324 mg/kg bw for rats (male/female); 
Dermal: Waiving, no test performed, substance is classified as higly corrosive according  to Directive 67/548/EEC, Annex I; 
Inhalation: LC50 = 2717 mg/m³ air for rats (male/female) after 4 h inhalation

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with acceptable restricions.

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Version / remarks:

Cited as Directive 84/449/EEC, B.1

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: strain Bor: WISW (SPF Cpb), Winkelmann, Borchen
- Age at study initiation: 8 wks (males) to 10 wks (females)
- Weight at study initiation: males 183 g mean; females 173 g mean (deviations from mean < 20 %)
- Fasting period before study: 16 hours before until 4 hours after dosing
- Housing: 5/group/cage
- Diet (e.g. ad libitum): Altromin 1324 pellets (standard diet; Altromin GmbH, Lage), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

paraffin oil

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw
- Justification for choice of vehicle: Test substance hydrolyses upon contact with water
- Lot/batch no. (if required): Merck-Schuchardt batch no. 706K3305274
- Purity: In order to increase stability of the test substance by elimination of water in the vehicle, the vehicle was dried at 100°C by introduction of nitrogen gas.


MAXIMUM DOSE VOLUME APPLIED: 5 ml/kg bw constant volume was used for application


DOSAGE PREPARATION (if unusual): preparation directly before dosing at room temperature

Doses:

20, 71, 133, 260, 310, 400 (only males), 500 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations several times on the day of dosing, twice daily on weekdays and once on weekends and bank holidays during the 14 d observation period; weighing was performed directly before dosing, after 1 week and at the end of the 14-d observation period
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

Calculation of LD50 according to Rosiello et al. (1977), J Tox Environ Health 3: 797, modified by Pauluhn (Bayer AG, report no. 11835, 1983); method based on the maximum-likelihood method according to Bliss (1938), Q J Pharm Pharmacol 11, 192.

Sex:

female

Dose descriptor:

LD50

Effect level:

270 mg/kg bw

Sex:

male

Dose descriptor:

LD50

Effect level:

377 mg/kg bw

Sex:

male/female

Dose descriptor:

LD50

Effect level:

324 mg/kg bw

95% CL:

283 - 371

Mortality:

male rats:
20 mg/kg: 0/5;
71 mg/kg: 0/5;
133 mg/kg: 0/5;
260 mg/kg: 0/5;
310 mg/kg: 1/5 (day 5);
400 mg/kg: 3/5 (within 2 days);
500 mg/kg: 5/5 (within 5 minutes)

female rats:
20 mg/kg: 0/5;
71 mg/kg: 0/5;
133 mg/kg: 0/5;
260 mg/kg: 2/5 (within 3 days);
310 mg/kg: 4/5 (within 2 days);
500 mg/kg: 5/5 (within 5 minutes)

Clinical signs:

Gasping, salivation, rough fur, sedation, poor condition, staggering gait, signs of aggressiveness in 2 males of the lowest dose group. Severity of symptoms demonstrated dose dependency, symptoms started 3 minutes after exposure and continued until day 7. During and immediately after application of 500 mg/kg bw the development of gases was observed from the stomach.

Body weight:

Body weight gain was retarded in male rats starting with 260 mg/kg bw and in female rats starting with 133 mg/kg bw.

Gross pathology:

Necropsy of intermittently deceased animals of the 500 mg/kg bw dose group demonstrated severe chemical burns in the gastrointestinal tract, and of the liver, kidneys and spleen. Upon necropsy a stinging odour was noted from the peritoneum. Deceased animals of the 260 mg/kg and higher dose groups showed changes of the gastrointestinal mucosa and/or discolouration of the liver.
Some of the animals sacrificed at the end of the observation period showed pale discolourations of the kidneys and/or swelling of the liver.

Other findings:

- Potential target organs: liver, kidney
- Other observations: One male of the 71 mg/kg bw dose group and one female of the 260 mg/kg bw dose group demonstrated alterations of the gastric mucosa.

During dosing and immediately thereafter development of gas was observed in the high dose group animals. Gross necropsy revealed corrosion of the gastrointestinal tract, liver, kidney and spleen in all animals that died during the study. During necropsy odor and gas development was reported.

Conclusion:

Based on the criteria of the DSD and the CLP criteria the test substance has to be considered as harmful if swallowed (R22, GHS Cat. 4).

Executive summary:

method: single orale application per gavage of test substance (dose 20, 71, 133, 260, 310, 400 (only males) and 500 mg/kg bw) to 5 male and 5 female rats, post-observation time 14 days

result: LD50 = 270 mg/kg bw (female rats) and LD50 = 377 mg/kg bw (male rats)

reference: Bomhard (Bayer AG), 1988

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

324 mg/kg bw

Quality of whole database:

The materials/methods and results are described in detail and are sufficient for evaluation.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline Study with acceptable restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Humidity of test atmosphere during exposure had to be reduced as the test substance hydrolyses in contact with humidity.

Principles of method if other than guideline:

Five male and five female Wistar rats were exposed to 5, 63, 403, 1326, 1596, 2544, 2832, 7986, 9318 mg thionyl chloride/m³ (vapour) for 4 hours. The animals were observed for mortality, weight and clinical signs through day 14. Exploratory lung function tests were conducted and a gross necropsy was performed.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen: Wistar Bor: WISW (SPF-Cpb)
- Age at study initiation: approx. 10 wks
- Weight at study initiation: 180 - 220 g mean weight
- Fasting period before study: not applicable, inhalation
- Housing: 5/cage
- Diet (e.g. ad libitum): Altromin 1324 - maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 1 wk


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

other: air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel inhalation chamber (dimensions: diameter = 30 cm, height = 28 cm) to which animal exposure tubes are attached, preventing contamination of the fur. Design of chamber prevents mixing of original test atmosphere with the exhalation air of the animals, which prevents the formation of hydrolysates caused by the humidity in the exhaled air.
- Exposure chamber volume: approx. 10 liter according to the authors (approx. 19.8 liters according to a calculation based on the reported dimensions of the chamber).
- Method of holding animals in test chamber: fixation of head and nose to the breathing zone by limitation of back off space in the exposure tubes.
- Source and rate of air: pressurized, filtered and dried air, 10.1 to 18.1 l/min, depending on required dilution in the respective dose groups
- Method of conditioning air: Pressurised air was generated by two compressors working in parallel, the generated air was dried and automatically conditioned by removing dust and oil, working pressure of 8 to 10 bar was reduced by reduction valves. The primary and dilution air used for the preparation of the test atmosphere were additionally dried. Ratio between supply and exhaust air was adjusted to ensure removal of 60 to 80 % of the supplied test atmosphere and a constant airflow towards the animals. These conditions ensured a constant air exchange, under comparable conditions a steady state is formed within 3 minutes (McFarland, 1976).
- System of generating vapours: 5 ml thionyl dichloride were filled into a small impinger tube kept at a temperature of 20°C by thermostatical control. Specified volmina of the gas atmosphere on top of the fluid were withdrawn by gas piston pumps via gas tight tubing (primary air), mixed with dilution air and directed into the inhalation chamber.
- Method of particle size determination: not applicable
- Treatment of exhaust air: remaining thionyl dichloride in the exhaust air was removed by reaction with NaOH in wash bottles.
- Temperature, humidity, pressure in air chamber: temperature and humidity were measured at the beginning, during and shortly before the end of exposure and a mean value calculated, air flow was constantly monitored throughout exposure. Temperature in the chamber ranged from 21 to 24°C, and relative humidity in the chamber exhaust air ranged from 17 to 44 % in the various exposure groups.


TEST ATMOSPHERE
- Brief description of analytical method used: nominal concentration was calculated from the weight difference of the impinger before and after the test, and the flow through of the inhalation chamber.
For analytical determination of the concentration the test substance was directed through an aqueous solution of sodium tetrachloromercurate. The resulting sulfur dioxide forms a dichlorosulfitemercurate complex which reacts with hydrogenchloride-discoloured pararosaniline and formaldehyde to a red-violet sulfonic acid. Determination of the concentration was performed by spectrophotometry at 560 nm (Eben, 1986).
- Samples taken from breathing zone: yes


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: not applicable, vapour
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): not applicable, vapour

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Methodology of analytical determination was reported separately (Eben, 1986); samples were taken from the breathing zone of the animals

Duration of exposure:

4 h

Concentrations:

analytical: 5, 63, 403, 1326, 1596, 2544, 2832, 7986, 9318 mg/m³ (corresponding to 0.005, 0.063, 0.403, 1.326, 1.596, 2.544, 2.832, 7.986, 9.318 mg/l)

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observation several times on the day of exposure, further schedule not specified; weighing was performed before exposure, and after 7 and 14 days; if technically applicable, lung function tests of all animals were performed on day 1 and 14 of the observation period in the 1596, 403 and 63 mg/m³ exposure groups.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, other: lung function (air flow, transpulmonary pressure, dynamic compliance, resistance, functional residual capacity, acetylcholine provocation test); procedures for determination of lung function were performed according to recommendations of Diamond and O'Donnell (1981), Likens and Mauderly (1979), Gross (1981) and Palacek (1969).

Statistics:

Calculation of LC50 according to Rosiello et al. (1977), modified by Pauluhn (1983). Procedure is based on the maximum-likelihood method according to Bliss, Q J Pharm Pharmacol (1938).

Sex:

male/female

Dose descriptor:

LC50

Effect level:

2.717 mg/L air

95% CL:

2.151 - 3.434

Exp. duration:

4 h

Remarks on result:

other: Corresponding to 2717 mg/m³ air.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

5.434 mg/L air

Exp. duration:

1 h

Remarks on result:

other: Calculated from 4 h value.

Sex:

male/female

Dose descriptor:

other: NOAEC (local effects)

Effect level:

0.005 mg/L air

Exp. duration:

4 h

Remarks on result:

other: Corresponding to 5 mg/m³ air. Respiratory irritation was observed at concentrations of 0.063 mg/mL (corresponding to 63 mg/m³) and above.

Mortality:

Males:
9.318 mg/l: 5/5 (within 3 h);
7.986 mg/l: 5/5 (within 3 to 4 h);
2.832 mg/l: 3/5 (within 4 h to 8 d);
2.544 mg/l: 3/5 (within 24 h to 2 d);
1.596 mg/l: 0/5;
1.326 mg/l: 1/5 (within 4 h);
0.403 mg/l: 0/5;
0.063 mg/l: 0/5;
0.005 mg/l: 0/5;

Females:
9.318 mg/l: 5/5 (within 2 h);
7.986 mg/l: 5/5 (within 3 h);
2.832 mg/l: 0/5;
2.544 mg/l: 4/5 (within 4h to 3 d);
1.596 mg/l: 0/5;
1.326 mg/l: 0/5;
0.403 mg/l: 0/5;
0.063 mg/l: 0/5;
0.005 mg/l: 0/5;

Clinical signs:

other: Number of animals with symptoms (and duration): 9.318 mg/l: 5/5 males and 5/5 females (until death); 7.986 mg/l: 5/5 males (1-4 h) and 5/5 females (1-3 h); 2.832 mg/l: 5/5 males (4 h-15 d) and 5/5 females (4 h-15 d); 2.544 mg/l: 5/5 males (4 h-15 d) an

Body weight:

Reduction of body weight gain beginning at 1.326 mg/l. In rats of the 0.403, 0.063 and 0.005 mg/l exposure groups the body weight gain was comparable to control animals.

Gross pathology:

Deceased animals: necrotic changes in the area of the nose and snout. Extremities showing a yellowish-greenish discolouration. Serous fluids were found in lung an thorax. Lungs were distended and showed liver-like changes. Liver appeared pale, with lobular pattern. Gastrointestinal tract reddened and filled with reddish mucus. Bleached incisives, appearing white.

Other findings:

- Other observations: local effects in the nose and the snout due to the irritating and corrosive character of the test substance.

Analysis of lung function revealed increased lung resistance at day 1 and day 14 after exposure in rats of the 1.596 mg/l exposure group, the authors concluded the occurrence of a progressive obstructive pulmonary damage, mostly impairing the upper and central airways. Strong breathing difficulties persisted during the whole observation period, the pathological investigations supported the indications for respiratory damage (lung edema, pleural effusion, chemical burns).

Rats of the 0.063 mg/l exposure group showed slightly reddened noses on the day of exposure, an impairment of breathing was not observed. From day 1 of the observation period these rats did not show any indications of adverse effects on breathing.

The vapours of thionyl dichlorid revealed a strong irritating potential to the respiratory tract. Due to the high water solubility the vapours mainly damage the upper respiratory tract.

A concentration of 5 mg thionyl dichloride/m³ air caused no clinical symptoms.

Executive summary:

method: similar to OECD TG 403 Nine groups of male and female Wistar rats, each containing 5 male and 5 female animals, were exposed (nose/head-only) once for 4 hour to different vapor atmospheres of thionylchloride (5, 63, 403, 1326, 1596, 2544, 2832, 7986, 9318 mg/m³). The animals were observed for 14 days after exposure. The vapors of thionyl dichlorid revealed a strong irritating potential to the respiratory tract. Due to the high water solubility the vapors damage the upper respiratory tract and no systemic toxicity is observed. LC50 (male + female rats) = 2717 mg/m³ air. A concentration of 5 mg thionyl dichloride/m³ air caused no clinical symptoms.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

2 717 mg/m³

Quality of whole database:

The materials/methods and results are described in detail and are sufficient for evaluation.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There was one study available on acute oral toxicity which was of good quality and followed an accepted guideline. Testing of dermal toxicity is not required according to Regulation (EC) No 1907/2006 due to the highly corrosive nature of the test substance. There were several studies available on acute inhalation toxicity, and one of them followed the guideline recommendations of a 4 -hour exposure period.

Overall, based on available reviews and handbooks, an experimental hydrolysis study in the gas phase and a recent guideline hydrolysis study according to OECD TG 111 “Hydrolysis as a Function of pH” it can be concluded that thionyl dichloride undergoes violent decomposition in aqueous milieu and decomposes quantitatively to HCl (CAS n° 7647-01-0) and SO2 (CAS n° 7446-09-5) with half-lives between 1.4 minutes and 5.9 minutes in the gas phase and < 2 minutes in water. SOCl2 is classified as R14 (Reacts violently with water). It is considered appropriate by the registrant to follow other thionyl dichloride evaluations and to conclude that due to spontaneous and exothermic hydrolysis thionly dichloride undergoes immediate disintegration. The data of the hydrolysis products, HCl and SO2, should be considered as appropriate and sufficient to characterize the toxicity of thionyl dichloride for endpoints where no data on the instable parent compound are available.

Evaluation of acute toxicity data after oral exposure:

Thionyl dichloride was investigated in an acute oral toxicity experiment and the oral LD50 was found to be 324 mg/kg (Bomhard 1988). Pale kidneys was reported during necropsy in surviving animals in all groups (20, 71, 133, 260, 310, 400 and 500 mg/kg; Bomhard 1988). “Pale” kidneys are frequently observed in rats probably due to intra-animal variations in blood flow and of unknown toxicological relevance since it is observed in all groups (no control group included) and in general without histopathologic correlation (no histopathology conducted in this study). Other “systemic” organs as liver and spleen are only affected in the high dose groups in animals that died due to corrosive effects on the gastrointestinal tract and should be considered as secondary effects based on the severe local toxicity leading to death. During dosing and immediately thereafter development of gas was observed in the high dose group animals. Gross necropsy revealed corrosion of the gastrointestinal tract, liver, kidney and spleen in all animals that died during the study. During necropsy odor and gas development was reported. Overall, this study indicates that thionyl dichloride is a primary corrosive compound and systemic organs are affected only at high doses where mortality occurred. The observations in this thionyl dichloride study are also consistent with data reported for HCl in which observations on liver, kidney and spleen were also mentioned in acute toxicity studies at lethal doses (e.g. MAK 2004, OECD SIDS 2002, ECB 2000, Hartzell et al., 1990, Darmer et al., 1974, MacEwean et al., 1974, Monsanto, 1976, Hoechst AG, 1966, Loewy and Munzer, 1923).

The LD50 value for thionyl dichloride is consistent with LD50 values observed for the hydrolysis product HCl (Hartzell et al., 1990, Darmer et al., 1974, MacEwean et al., 1974, Monsanto, 1976, Hoechst AG, 1966, Loewy and Munzer, 1923) cited in OECD SIDS for HCl and ECB 2000. The German “Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area” (MAK) reported LD50 of about 237 mg/kg for HCl (e.g. MAK 2004, see HCl review and key study).

For SO2 no acute oral toxicity studies are available (gaseous substance), but LC50 values after inhalation exposure are in the same range as LC50 values for thionyl dichloride (e.g. Pauluhn 1991, RTECS, JCTODH 1977, MAK 1998, TRGS900, 2011).

Thionyl dichloride and both hydrolysis products are considered to be corrosive; thionyl dichloride is labeled with R35 (causes severe burns). The available data on thionyl dichloride, HCl and SO2 show that local effects (corrosion of the gastrointestinal tract) is the primary effect of these compounds and systemic organs are affected only at high doses where mortality occurred.

Evaluation of acute toxicity data after dermal exposure:

Local effects: Thionyl dichloride and both hydrolysis products are considered to be corrosive; thionyl dichloride is labeled with R35 (causes severe burns).

Evaluation of acute toxicity data after exposure via inhalation::

There is a reliable thionyl dichloride study available conducted according to OECD guideline 403, well documented, and the concentrations applied had been analytically confirmed and were sufficient to derive an 4-hour LC50. Therefore, it was chosen as key study. The other inhalation studies available for rats were also of good quality but included only 1-hour exposure periods. Nine groups of male and female Wistar rats, each containing 5 male and 5 female animals, were exposed (nose/head-only) once for 4 hour to different vapor atmospheres of thionyl dichloride (5, 63, 403, 1326, 1596, 2544, 2832, 7986, 9318 mg/m³). The animals were observed for 14 days after exposure. The vapors of thionyl dichlorid revealed a strong irritating potential to the respiratory tract. Due to the high water solubility the vapors damage the upper respiratory tract and no systemic toxicity is observed. LC50 (male + female rats) = 2717 mg/m³ air. A concentration of 5 mg thionyl dichloride/m³ air caused no clinical symptoms.

Additionally, an old inhalation study for thionyl dichloride in cats was cited in a handbook (1931) which reported an LC100 of (probably) 0.085 g/l air after 20 minutes of exposure. Due to a mistake in conversion from one concentration unit to the other, the actual concentration tested was equivocal, a conversion of 0.085 g/l to 17.5 ppm (which is equivalent to 0.085 mg/l) was given. No further information was reported. Further, this result is discussed in the study by Kinkead and Einhaus and considered to be not reliable. Therefore, this study is considered to be unreliable for risk assessment.

The acute oral LD50 values for HCl were determined to be 238-277 mg/kg bw for female rats, and the inhalation LC50 values were determined to be 23.7-60.9 mg/L/5min, 5.7-7.0 mg/L/30min and 4.2-4.7 mg/L/60min for rats, 20.9 mg/L/5min, 3.9 mg/L/30min and 1.7 mg/L/30min for mice. Hydrogen chloride is corrosive to the skin and severe effects can be expected from exposure to the eyes (OECD SIDS for HCl).

In an acute inhalation study with SO2 (Pauluhn 1991) in rats a LC50 = 1399 ppm (= 3721 mg SO2/m³air) for an exposure of 30 min was determined. The rats revealed a concentration dependant irritation of the respiratory system with long lasting breathing difficulties and a local eye damage (opacity of the cornea). The delayed mortality is regarded as caused by lung damage due to irritation/corrosion.

No observation on any systemic organ was observed in acute inhalation studies with thionyl dichloride (Pauluhn 1987 and 1986), HCl (e.g. MAK 2004, ECB 2000) or SO2 (e.g. MAK 1998, JCTODH, 1977). The local irritation potency of thionyl dichloride, HCl and SO2 are similar leading to comparable LC50 values in agreement with immediate disintegration. Thionyl dichloride: LC50 (1h, Pauluhn 1986): 5434 mg/l, HCl LC50 (1h, MAK 2004): 2810-3120 ml/m3 (Hartzell 1990), SO2: LC50 (30 min) = 1399 ppm (ca. 3721 mg/m³Pauluhn(1991) to 3000 ppm (ca. 7980 mg/m³)(JCTODH, 1977).

In conclusion, the LD50 and LC50 values of thionyl dichloride are in the same range as for HCl and SO2. The clinical and pathological findings of thionyl dichloride, HCl and SO2 are similar and consistent with local corrosively at the port of entry.

Justification for selection of acute toxicity – oral endpoint
A valid acute oral toxicity study for thionyl chloride (SOCl2) is available.
SOCl2 is hydrolyzed rapidly and completely by water in an exothermic reaction with formation of HCl and SO2. Therfore the toxicity of the hydroysis products is also considered.

Justification for selection of acute toxicity – inhalation endpoint
Several acute inhalation toxicity studies for thionyl chloride (SOCl2) are available.
SOCl2 is hydrolyzed rapidly and completely by water in an exothermic reaction with formation of HCl and SO2. Therfore the toxicity of the hydroysis products is also considered.

Justification for classification or non-classification

According to the criteria of the DSD and the criteria of the CLP regulation the test substance (SOCl2) has to be classified as:

R22, Cat. 4: harmful if swallowed

R20: harmful if inhaled; Cat. 3: toxic if inhaled