N,N'-dimethyldiphenylthiuram disulphide

Endpoint summary

Description of key information

There is no subchronic study available. 
In the  available  28-day gavage study with rats (0, 100, 300 and 1000 mg/kg bw/day  given as suspension  in  Solutol 15 HS /Ethanol /water  =  4 : 1 : 5, Wirnitzer 2012, draft report) N,N-Dimethyldiphenylthiuram  disulphide caused significant hemolytic anemia from 100 mg/kg bw/day (LOAEL) onwards increasing in severity with the dose accompanied by  histopathological  effects in spleen and kidney but not in the liver. Based on these results a 90-day oral study in rats according OECD TG 408 and GLP is proposed

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study and GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks
- Weight at study initiation: males 152.4 g; females: 137.0 g
- Housing: in groups with 3 rats
- Diet ad libitum
- Water ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 55
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Solutol HS 15 / Ethanol / Tap Water = 4 : 1 : 5

Details on oral exposure:

The test item was administered by gavage in vehicle. the administration volume 10 ml/kg bw per day. The formulations were prepared as needed taking into account the analytically determined stability. The test item was administered as a suspension in vehicle.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

dose levels were selected based on results of a previous dose range finding study with gavage administration in rats in the same laboratory
study design as respective guideline

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes ,
twice a day for morbidity and mortality
daily for general clinical findings
BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION weekly


WATER CONSUMPTION weekly

HAEMATOLOGY: Yes , day 29
- Parameters checked : differential blood countm erythr9ocyte morphologym erythrocyte countm mean corcuscular hemoglobin, mean corpuscular hemoglobinconcentrationm mean corpuscular volume, hemoglobin concentration, hematocrit, leukocyte countm reticulocyte countm thrombocyte count, thromboplastin time (Hepato Quick)

CLINICAL CHEMISTRY: Yes , day 29
- Parameters checked: glucose concentration, alanine aminotransferase, aspartate amino transferase, albumin, total bilirubin, cholesterol, creatinine, total protein, urea, glucose, potassium, sodium, gall acids


NEUROBEHAVIOURAL EXAMINATION: Yes
functional observation battery day 26
Motor/Locomotor Activity day 26:

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
organ weights: brain, heart, liver, spleen, kidneys (both), thymus, adernal glands(both), prostate, seminal vesicle,with coagulation glands, epididymides (both), testes(both), ovaries(oviduct(both) and uterus/cervix
Histopathology

Other examinations:

no data

Statistics:

Dunnett Exact Homogeneous Test, Dunnett exact Homogeneous test after logarithmic transformation, Bonferroni/Mann-Whitney U-test

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
-survival was not affected
- the only clinical observation was discolored feced at mid and high dose rats (both sexes)

BODY WEIGHT AND WEIGHT GAIN
mean body weight development was slightly affected in high dose animals:
Compared to controls over the period of treatment body weight developpment was reduced in high dose males by 9 % and in high dose females by 12 %

HAEMATOLOGY
at all dose levels mostly significantly reduced erythrocyte count, hematocrit, hemoglobin concenteration and MCHC were seen associated with increased MCH and MCV as well as increased reticulocyte counts (see table in section Any other information on results including tables)
Macrocytosis was seen in all female dose groups and in mid and high dose males, in the later group with increased grading
Platelet counts and thromboplastin time (HQuick) were unchanged in the whole dose range tested.
White blood counts in all treated rats were comparable to concurrent controls

CLINICAL CHEMISTRY
Bilirubin concentration wa increased in all dose levels
slgnificantly in 1000 mg /kg bw/day females: 1.18 µmol/l (p<=0.05) versus 0.34 µmol/l in controls
significantly in males: 100, 300, 1000 mg/kg bw versus control:
1.04 µmol/l (p<=0.05), 1.08µmol/l (p<=0.05), 1.92µmol/l (p<=0.05) versus 0.15µmol/l

GROSS PATHOLOGY
ORGAN WEIGHTS
Spleen
Only mean spleen weights(absolut and relative) were mostly significantly and dose related increased in all dose grouppppps (both sexes):
100, 300, 1000 mg/kg bw versus control ( ++ = p<=0.01)
male, absolute : 1030.4++, 1222.2++, 1560.0++ versus 568.8
male, relative: 0.3521++, 0.4391++, 0.5820++ versus 0.2023
female, absolute:699.2, 816,8+, 1040.2++ versus 568.8
female, relative: 0.3514, 0.4082++, 0.5612++ versus 0.2659

all other organ weights were inconspiduous

HISTOPATHOLOGY: NON-NEOPLASTIC
MALE
---1000 mg/kg bw/day
SPLEEN:
increased extramedullary hematopoiesis and sinus congestion,
increased capsular thickening,
increased pigment deposition, marginal zone atrophy
FEMORAL BONE MARROW
increased cellularity
KIDNEYS
tubular swelling
---300 mg/kg bw/day
SPLEEN
increased extramedullary hematopoiesis and sinus congestion,
increased capsular thickening
KIDNEYS
tubular swelling
---100 mg/kg bw/day
SPLEEN
increased extramedullary hematopoiesis and sinus congestion
KIDNEYS
tubular swelling

FEMALES
---1000 mg/kg bw/day:
SPLEEN:
increased extramedullary hematopoiesis and sinus congestion, increased pigment deposition
FEMORAL BONE MARROW
increased cellularity
KIDNEYS
tubular swelling, , pigment deposition
---300 mg/kg bw/day:
SPLEEN:
increased extramedullary hematopoiesis and sinus congestion, increased pigment deposition
---100 mg/kg bw/day:
SPLEEN:
increased extramedullary hematopoiesis and sinus congestion, increased pigment deposition

There were no test substance related gross and/or histopathological changes beyond those described.

Dose descriptor:

LOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: based on effects on hematology, spleen and kidneys

Critical effects observed:

not specified

Hematology

dose(mg/kg bw)	Ery(T/L)	HB(g/L)	HCT(L/L)	MCH(pg)	MCHC(g/LEry)	MCV(fL)	Reti (%)	Thro(g/L)
males
0	8.518	159	0.5144	18.72	309.6	60.38	2.32	728.0
100	7.262 ++	141.8 ++	0.4728 ++	19.56	300.2+	65.20+	6.92+	833.0
300	7.182 ++	145.2 ++	0.4862	20.24 ++	298.6+	67.70 ++	9.46+	836.4
1000	6.204 ++	135.8 ++	0.4522 ++	21.90 ++	300.2+	72.98 ++	11.66+	886.2
females
0	7.980	148.4	0.4618	18.62	321.6	57.86	2.64	822.2
100	6.574 ++	132.2 ++	0.4220 +	20.12 ++	313.0 ++	64.34 ++	7.50+	852.0
300	6.192 ++	129.2 ++	0.4162+	20.92 ++	310.6 ++	67.26 ++	10.64+	888.6
1000	5.714 ++	123.8 ++	0.3946 ++	21.68 ++	313.6 ++	69.12 ++	11.92+	847.6

+ = p<=0.05 ++ = p<=0.01

Executive summary:

To examine N,N'-dimethylphenylthiuram disulfide for subacute oral toxicity according to OECD TG 407 and GLP, male and female Wistar rats received 0, 100, 300 and 1000 mg/kg bw/day dissolved in Solutol HS 15 / Ethanol / tap water (4 : 1 : 5) over a period of 28 days.

Survival was not affected by treatment. The only clinical observation was discolored feced at mid and high dose rats (both sexes). Compared to controls over the period of treatment, body weight development was reduced in high dose males by 9 % and in high dose females by 12 %. Hemolytic anemia is derived from reduced erythrocyte count, hematocrit, hemoglobin concentration and MCHC, increased MCH, MCV, reticulocyte counts (all dose levels) and macrocytosis (low dose females, both sexes at mid and high dose), increased bilirubin concentration (all male dose groups, 1000 mg/kg-females), increased spleen weights (both sexes all dose groups) and histopathological findings in spleen [increased extramedullary hematopoiesis and sinus congestion (all dose groups both sexes), increased pigment deposition (females all dose groups, males 1000 mg/kg), increased capsular thickening (males 300 and 1000 mg/kg), marginal zone atrophy (males 1000 mg/kg)]. In kidneys tubular swelling (males all dose groups, females 1000 mg/kg bw/day and pigment deposition (females 1000 mg/kg bw/day) were observed. Secondarily femoral cellularity was increased (both sexes, only high dose investigated).

Thus, under the conditions described, a no-observed-effect-level (NOAEL) for N,N'-dimethyldiphenylthiuram disluphide could not be established, the LOAEL is 100 mg/kg bw/day.

.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and has Klimisch score 1.
There is no sub chronic study available. as required by Regulation (EC)1907/2006 (REACH) ANNEX IX section 8.6.2 There is a reliable short term toxicity study (28 days) which was performed according to OECD TG 407 and GLP available showing severe toxicity effects which fulfill the criteria for classifying the substance as R48 respective to allocate the substance to STOT Re category 2 according to Regulation (EC) 1272/2008 (GHS-CLP). This available short term study does not provide a NOAEL but a LOAEL.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no sub chronic study available. as required by Regulation (EC)1907/2006 (REACH) ANNEX IX section 8.6.2

To examine N,N'-dimethylphenylthiuram disulfide for subacute oral toxicity according to OECD TG 407 and GLP, male and female Wistar rats received 0, 100, 300 and 1000 mg/kg bw/day dissolved in Solutol HS 15 / Ethanol / tap water (4 : 1 : 5) over a period of 28 days (OECD TG 407 and GLP).

Survival was not affected by treatment. The only clinical observation was discolored feced at mid and high dose rats (both sexes). Compared to controls over the period of treatment, body weight development was reduced in high dose males by 9 % and in high dose females by 12 %. Hemolytic anemia is derived from reduced erythrocyte count, hematocrit, hemoglobin concentration and MCHC, increased MCH, MCV, reticulocyte counts (all dose levels) and macrocytosis (low dose females, both sexes at mid and high dose), increased bilirubin concentration (all male dose groups, 1000 mg/kg-females), increased spleen weights (both sexes all dose groups) and histopathological findings in spleen [increased extramedullary hematopoiesis and sinus congestion (all dose groups both sexes), increased pigment deposition (females all dose groups, males 1000 mg/kg), increased capsular thickening (males 300 and 1000 mg/kg), marginal zone atrophy (males 1000 mg/kg)]. In kidneys tubular swelling (males all dose groups, females 1000 mg/kg bw/day and pigment deposition (females 1000 mg/kg bw/day) were observed. Secondarily femoral cellularity was increased (both sexes, only high dose investigated).

Thus, under the conditions descriibed, a no-observed-effect-level (NOAEL) for N,N'-dimethyldiphenylthiuram disluphide could not be established, the LOAEL is 100 mg/kg bw/day.

Overall conclusion

There is no sub chronic study available. as required by Regulation (EC)1907/2006 (REACH) ANNEX IX section 8.6.2 There is a reliable short term toxicity study (28 days) which was performed according to OECD TG 407 and GLP available showing severe toxicity effects which fulfill the criteria for classifying the substance as R48 (DSD-DPD) respective to allocate the substance to STOT Re category 2 according to Regulation (EC) 1272/2008 (GHS-CLP). This available short term study does not provide a NOAEL but a LOAEL.

Hemolytic anemia with accompanied by histopathological effects in spleen and kidney are observed at all doses tested in the sub-acute toxicity study. Hemolytic anemia that accompany methaemoglobinaemia or haemolysis is considered to be an acute effect (e.g. see The MAK-Collection Part I: MAK Value Documentations, Aniline, Vol. 26. DFG, 2011). Since there are fundamental physiological differences between rats and humans and disorders observed in humans that accompany methaemoglobinaemia or haemolysis, is not expected that these effects occur in human spleens. Based on the effects observed a test proposal is included in the dossier for a 90-day oral study in rats according to OECD TG 408. In the meantime a provisional DNEL is calculated taking into account a time extrapolation factor of 2. This factor will be reconsidered once the data from the 90 day study become available.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
only study available:
Guideline study and GLP

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: hematopoiesis

Justification for classification or non-classification

There is a reliable short term toxicity study (28 days) which was performed according to OECD TG 407 and GLP available showing severe toxicity effects which fulfill the criteria for classifying the substance as R48 (DSD-DPD) respective to allocate the substance to STOT Re category 2 according to Regulation (EC) 1272/2008 (GHS-CLP).