Registration Dossier

Administrative data

Description of key information

- In this GLP complaint oral repeated dose toxicity study, performed according to OECD 408, the no-observed-adverse-effect level (NOAEL) is conservatively placed at the lowest level tested (10 mg/kg body weight/day) based on increased liver and kidney weight in combination with effects on total protein and albumin in the mid-dose group (males).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar 2018 to 26 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 21st September 1998.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21.8.2001
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar outbred rats (Crl:WI(Han)), (SPF).
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
The Han rat strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 5 to 6 weeks.
- Weight at study initiation: For males: 121 to 152 g (mean 136 g); for females: 97 to 137 g (mean 117 g).
- Housing: The animals were housed under conventional conditions in one room. No other animals were housed in the same room during the study. The animals were kept in macrolon cages with wood shavings (Lignocel) as bedding material, and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex.
- Diet: Cereal-based (closed formula) rodent diet (VRF1(FG)), ad libitum unless precluded by the performance of certain laboratory investigations
- Water: Tap water, ad libitum unless precluded by the performance of certain laboratory investigations
- Acclimation period: Quarantine period: 14 March to 19 March. Study commenced on 26 March.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet was analysed by the supplier for nutrients and contaminants. Routine physical, chemical and microbiological examination of drinking water was conducted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65 except during brief periods associated with room cleaning
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
26 Mar 2018 to 26 Jun 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
On 22 and 23 March 2018, the appropriate amount of test substance was weighed into a glass bottle for each day of the study and for each dosing group. The vials were stored at ambient temperature. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred on a magnetic stirrer for at least 30 minutes, until visual homogeneity was obtained. All suspensions were continuously stirred throughout the dosing procedure, in order to maintain the homogeneity of the test substance in the vehicle. The concentration of the test substance were 2, 10 and 50 mg/mL for the low-dose, mid-dose and high-dose, respectively.

- VEHICLE
- Amount of vehicle: 5 mL/kg bw/day
- Batch no.: A1701528
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of the test substance in the gavage suspension was conducted using ICP-MS. The presence of the test substance in the test dilutions was determined by analysis of the Zinc content. The Zinc content was assumed to be proportional to the concentration of the test substance. The analyses were performed by inductively coupled plasma mass spectrometry (ICP-MS) after digestion with sulphuric acid and hydrogen peroxide.
Because the method involves the determination of the Zinc concentration only, the stability of the test substance could not be established. Therefore, fresh test dilutions were prepared daily.
The homogeneity (and content) of the test substance in the test dilutions were demonstrated in one batch prepared on 29 March 2018, by analysing three samples (taken at top-, mid- and bottom- of the vial) of each test dilution. The content of the test substance in each test dilution was determined in the batches prepared on 29 March, 23 April, 22 May and 18 June 2018.
Duration of treatment / exposure:
13 consecutive weeks.
Frequency of treatment:
Daily, seven days a week.
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Group 2, low-dose.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 3, mid-dose.
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 4, high-dose.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the sponsor and were based on the results of a dose-range finding study (DRF; V21151/01) as well as several earlier studies with the test substance or the structural analogue Zinc bis(diethyldithiocarbamate) (ZDEC). In the DRF, statistically significant increases were seen for the kidney weights in mid-dose and high-dose males (with a comparable but non-statistically significant increase in females) and for the liver weights in high-dose females. This is in good agreement with the observations that were described by Gray (1978).
- Rationale for animal assignment (if not random): Males and females were allocated to experimental groups proportionately to body weight by computer randomization (the animals of which the body weight deviated most from the mean were automatically eliminated).
- Rationale for selecting satellite groups: Surplus animals (3 males and 3 females) were kept in the animal room as sentinel animals and used for training purposes after termination of the study.
- Section schedule rationale (if not random): Day 91 and day 92 for males and females, respectively.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in the morning and additionally in the afternoon for dead or moribund animals to minimize loss of animals from the study.
- All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: If necessary, handled to detect signs of toxicity daily in the morning. In addition all rats were subjected to detailed clinical observations prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Day -3, 0 and subsequently once weekly. The animals were weighed on their scheduled necropsy date.

FOOD CONSUMPTION: Yes
- Food consumption was measured per cage by weighing the feeders. The consumption was measured over one-week periods throughout the treatment period for all animals in the cage. The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Water consumption was measured per cage, by weighing the drinking bottles daily, during 5-day periods in or about weeks 1, 6 and 11. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day -5 (all males), day -4 (all females), and in all rats of the control group and the high-dose group in week 13 (day 87).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight (water was freely available).
- How many animals: All surviving animals.
- Parameters examined: haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), Reticulocytes, total white blood cell count (WBC), differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time, thrombocyte count (platelet count).
- The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight (water was freely available).
- How many animals: All surviving animals.
- Parameters examined: alkaline phosphatase activity (ALP), fasting glucose, aspartate aminotransferase activity (ASAT), bilirubin total, alanine aminotransferase activity (ALAT), cholesterol, gamma glutamyl transferase activity (GGT), triglycerides, total protein calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate, Thyroxin (T4).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for 16 hours (water was freely available).
- How many animals: All surviving animals.
- Parameters examined: volume, occult blood, density (specific gravity), ketones, appearance (colour and clarity), protein, pH, bilirubin, glucose, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the study.
- Dose groups that were examined: All rats.
- Battery of functions tested: Functional Observation Battery and motor activity assessment (week 12)

IMMUNOLOGY: No

OTHER: ANALYSIS OF THYROID HORMONES (T4 AND TSH)
- Time schedule for collection of blood: At necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight (water was freely available).
- How many animals: All surviving animals.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The following organs were weighed: adrenals, prostate, brain, seminal, vesicles, (with coagulating glands), epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroids, ovaries, uterus.
- The following tissues were preserved: adrenals, oviducts (=fallopian tubes), aorta, pancreas, axillary lymph nodes, parathyroid, brain, parotid salivary glands, cecum, pituitary, colon, prostate, duodenum, rectum, epididymides, seminal vesicles and coagulating glands, oesophagus, skeletal muscle (thigh), exorbital lachrymal glands*, skin (flank), eyes, spinal cord, femur with joint*, spleen, GALT (gut associated lymphoid tissue, including Peyer's patches), sternum with bone marrow, stomach, heart, sublingual salivary glands, ileum, submaxillary salivary glands, jejunum, testes, kidneys, thymus, liver, thyroid, lungs, trachea/bronchi, mammary gland, (females), urinary bladder, mandibular (cervical) lymph nodes*, uterus (with cervix), mesenteric lymph nodes, vagina, nerve-peripheral (sciatic), all gross lesions, ovaries.
* The tissues marked with * were preserved but not processed for histopathological examination, unless histopathological examination was considered necessary on the basis of gross observations.

HISTOPATHOLOGY: Yes
The tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) were performed on all tissues and organs listed above - except those marked with an asterisk - of all animals of the control group and the high-dose group. Gross lesions were examined in rats of all dose groups.
Other examinations:
THYROID HORMONES
Thyroid Stimulating Hormone (TSH) levels were assessed at necropsy in serum using commercially available enzyme-linked immunosorbent assays (ELISAs). Thyroxine (T4) was analysed in plasma collected at necropsy using the Siemens Dimension ExL
Statistics:
A list of the statistical test performed can be found in Table 1 and 2 in 'Any other information on materials and methods incl. tables'.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was occasionally noted in three males and three females of the high-dose group and, at one occasion, in one mid-dose male. These findings may be related to the dosing procedure (oral gavage), but are not considered adverse. The few other findings noted comprised random observations such as sparsely haired areas, encrustation(s) or wounds of the skin, mouth and tail and tail tip missing or a kink in the tail, discharge of the eye(s) or broken upper incisors. Because of the incidental occurrence of these findings, they are not ascribed to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Table 1 and 2 in 'Any other information on results incl. tables'.
Mean body weights were somewhat (≤ 9%) reduced in males of the high-dose group. The difference with the controls attained statistical significance in weeks 5, 6 and 13. Also the terminal body weights were statistically significantly decreased (about 11%) in high-dose males.
Body weight gain was statistically significantly reduced in high-dose males at various stages, including the overall body weight gain (day 0-90). Only incidental statistically significant differences from control values occurred in males and females of the other dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake (cage means) was not affected by the treatment. At one occasion (week 4), food intake was slightly, though statistically significantly decreased in high-dose females, but there were no significant differences in overall food intake among the groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Overall water consumption tended to be slightly elevated in males of the mid- and high-dose groups in weeks 6 and 11, but there were no statistically significant differences in water consumption (cage means) among the groups, apart from an incidental decrease in high-dose females on day 39-40.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmoscopic examination in week 13 did not reveal any treatment-related changes. The few findings noted occurred only incidentally.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Table 3 to 6 in 'Any other information on results incl. tables'.
Mean corpuscular volume (MCV) was statistically significantly decreased and mean corpuscular haemoglobulin concentration (MCHC) was increased in both males and females of the high-dose group. The changes were only very slight (2-5%) and the main parameters from which these calculated values were derived were not significantly affected. Therefore they are not considered to represent an adverse effect.
Thrombocytes were statistically significantly increased in high-dose females and relatively high in males. The percentage of lymphocytes was statistically significantly decreased and the percentage of neutrophils was increased in high-dose males. The values were within the range of historical control data. Because, moreover, these findings were not reflected in significant differences in the absolute lymphocytes and neutrophils counts, no significance is attached to these changes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Table 7 and 8 in 'Any other information on results incl. tables'.
Total protein and albumin concentrations showed a dose-related decrease in males of the mid- and high-dose groups, while the A/G ratio was decreased in high-dose males. In females, albumin concentration and A/G ratio were decreased in the high-dose group.
Inorganic Phosphate concentration was increased in the high-dose group in both sexes.
Chloride concentration was decreased in high-dose females; the change was within the range of historical control data.
ASAT activity was slightly increased in high-dose males; the change was within the range of historical control data.
ALAT activity was reduced in mid- and high-dose females and in mid-dose males. These findings were in the range of historical control data and are not considered to be relevant because an increase rather than a decrease in this parameter is considered the reflection of a toxic effect and because the differences with the controls were only slight.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The urinary volume was statistically significantly increased in high-dose males. The urinary density was not affected.
Semi-quantitative (dipstick) urinary measurements showed a significantly increase in the incidence of urinary ketones in females of the high dose group. This increase was mainly due to three females that showed approximate values between 4 and 8 mmol/L (=gradation 2). This severity is generally not observed in female animals.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The results of the weekly detailed clinical observations, neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of ZDBC in rats. However, a number of treatment-related effects were observed in animals of the mid- and high-dose groups. These effects were observed during the weekly detailed clinical examinations and/or during the functional observational battery testing at the end of the study period.
In the high-dose group these effects included sliding with the ventral parts of the head and neck over the bottom of the open field, piloerection, salivation, occasional hypoactivity, and, in males only, decreased hindlimb grip strength and increased body temperature. Effects observed in the mid-dose group included piloerection and, in males only, salivation and a decrease in hindlimb grip strength.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Table 9 to 13 in 'Any other information on results incl. tables'.
The following statistically significant differences with the controls were noted in organ weights:
- The relative weight of the liver was increased in the mid- and high-dose group in both sexes. The absolute weight of this organ was increased in females of the mid- and high-dose group and in males of the high-dose group.
- The relative weight of the kidneys was increased in males of the mid- and high-dose group and in females of the high-dose group. The absolute weight of the kidneys was increased in high-dose males.
- The absolute and relative weights of the spleen were increased in high-dose females.
- The relative weight of the thymus was decreased in high-dose females and relatively low in high-dose males (not statistically significant in males). The absolute weight of this organ was decreased in males and female of the high-dose group.
- The relative weight of the adrenals was increased in high-dose males.

Other statistically significant changes in organ weighs were noted that are considered not to be directly related to the treatment:
- The relative weight of the testis was increased in the mid-and high-dose group. The absolute testes weight was not affected. This finding is ascribed to the lower terminal body weights in these groups, most evident in the high-dose group. During growth retardation, rats tend to maintain their (absolute) weight of the testes, and there is a well-known inverse correlation between terminal body weight and relative testes weight.
- The absolute weights of the brain and heart were slightly decreased in high-dose males. This was probably related to the lower terminal body weights in this group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy no treatment-related macroscopic changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic evaluation did not reveal treatment-related histopathological changes. The histopathological changes observed were about equally distributed between the high-dose group and the controls or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
ANALYSIS OF THYROID HORMONES
Thyroid Stimulating Hormone (TSH) and Thyroxine(T4) levels, as assessed in serum using ELISA (Historical data can be found in Table 14 and 15 in 'Any other information on results incl. tables'). There were no statistically significant or dose-related changes in TSH or T4 levels. By mistake, T4 hormone levels were also analyzed in plasma using the Siemens Dimension ExL, Clinical Chemistry system. The results of this analysis showed a statistically significant decrease in T4 levels in high-dose females.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Analysis of the dosing dilutions

The analytical report confirms the correct preparation of the dosing dilutions and concludes that the test substance was homogeneously distributed in the carrier at each test concentration. The concentration of the test substance was close to intended (94-107%) at all dose levels for all gavage liquids analysed. Therefore, it was concluded that the animals received the intended concentration of test substance at all dose levels.

Table 1. Body weight males - Day(s) Relative to Start Date

Sex: Male

Bodyweights

 Bwday -x

(g)

 

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

-3

0

7

14

21

28

35

42

49

56

63

70

77

84

90

0 mg/kg

Mean

SD

N

116.03

6.30

10

135.90

7.01

10

179.10

10.20

10

222.08

10.98

10

260.29

12.34

10

284.10

14.15

10

305.68

11.50

10

324.19

13.52

10

336.55

14.51

10

348.68

16.32

10

358.80

18.45

10

368.28

18.77

10

375.98

18.02

10

381.00

18.54

10

385.94

18.52

10

10 mg/kg

Mean

SD

N

116.69

8.49

10

135.58

7.98

10

177.04

9.59

10

219.08

12.09

10

254.60

13.26

10

276.31

15.34

10

296.58

16.58

10

312.98

20.73

10

325.58

22.22

10

336.48

24.10

10

345.59

24.84

10

353.93

26.32

10

363.25

27.49

10

370.10

28.41

10

372.41

27.79

10

50 mg/kg

Mean

SD

N

116.63

8.24

10

135.95

8.76

10

176.02

11.50

10

216.25

15.28

10

249.91

16.21

10

274.14

18.14

10

298.95

20.75

10

315.63

21.57

10

329.65

23.97

10

340.66

26.40

10

347.79

27.58

10

355.29

33.05

10

365.11

30.92

10

372.97

31.44

10

373.23

31.23

10

250 mg/kg

Mean

SD

N

117.53

7.90

10

137.69

7.32

10

175.43

10.48

10

214.04

10.94

10

244.82

13.32

10

265.24

14.35

10

282.12 ** 14.44

10

298.78**

15.82

10

311.14

18.65

10

321.36

21.42

10

328.54

22.69

10

336.21

21.74

10

344.27

24.01

10

352.11

25.75

10

350.78 *

24.69

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01


Table 2. Body weight females - Day(s) Relative to Start Date

Sex: Female

Bodyweights

 Bwday -x

(g)

 

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

  Bw (g)

[G]

-3

0

7

14

21

28

35

42

49

56

63

70

77

84

90

0 mg/kg

Mean

SD

N

105.89

8.09

10

118.41

7.78

10

141.81

9.39

10

156.67

9.52

10

168.46

10.91

10

180.05

13.31

10

187.46

14.55

10

196.71

15.39

10

198.28

17.19

10

203.80

15.72

10

205.26

15.94

10

208.22

15.93

10

210.36

15.63

10

213.40

15.44

10

216.81

13.45

10

10 mg/kg

Mean

SD

N

105.68

7.82

10

117.37

6.41

10

138.96

5.98

10

154.71

7.66

10

170.96

8.63

10

179.38

12.85

10

190.11

11.21

10

196.19

14.33

10

203.22

14.37

10

205.69

16.60

10

209.08

15.42

10

211.39

16.36

10

213.19

16.37

10

215.16

17.79

10

218.19

17.43

10

50 mg/kg

Mean

SD

N

105.03

8.60

10

115.70

5.87

10

137.52

6.13

10

150.43

8.16

10

164.51

8.21

10

173.65

8.76

10

184.29

6.67

10

190.01

8.44

10

195.99

10.57

10

199.46

9.40

10

203.41

7.70

10

205.88

9.86

10

207.58

12.09

10

208.88

11.48

10

210.67

11.04

10

250 mg/kg

Mean

SD

N

105.29

9.19

10

116.20

8.68

10

137.57

10.79

10

151.13

12.26

10

165.05

13.55

10

173.63

15.26

10

182.62

15.32

10

190.00

16.01

10

195.00

16.66

10

199.22

16.88

10

202.67

17.73

10

204.62

17.80

10

206.35

18.62

10

207.40

19.02

10

209.72

18.41

10

[G] - Ancova/Anova & Dunnett

 

Table 3. Red blood cell and coagulation parameters - Day 91 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

[G]

Hb (mmol/L)

[G1]

PCV (L/L)

[G1]

MCV (fL)

[G1]

MCH

(fmol)

 

[G1]

MCHC

(mmol/L)

 

[G1]

Reticulo cytes

(%)

 [G2]

Thrombo cytes

(10E9/L)

 [G2]

Prothrom Time

(s)

 [G1]

0 mg/kg

Mean SD

N

9.061

0.306

10

9.64

0.31

10

0.5013

0.0194

10

55.34

1.69

10

1.064

0.035

10

19.24

0.26

10

2.043

0.178

10

801.5

54.1

10

20.23

1.33

10

10 mg/kg

Mean SD

N

9.235

0.418

10

9.95

0.33

10

0.5148

0.0140

10

55.79

1.22

10

1.078

0.029

10

19.33

0.40

10

2.022

0.145

10

806.2

98.7

10

18.91

1.68

10

50 mg/kg

Mean

SD

N

9.358

0.396

10

9.92

0.38

10

0.5116

0.0203

10

54.69

1.16

10

1.060

0.022

10

19.39

0.31

10

2.007

0.316

10

823.8

62.7

10

17.56

2.77

10

250 mg/kg

Mean SD

N

9.186

0.550

10

9.66

0.44

10

0.4906

0.0227

10

53.48 * 2.00

10

1.053

0.050

10

19.69 ** 0.29

10

2.026

0.348

10

895.5

103.4

10

18.30

2.48

9

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 4. Red blood cell and coagulation parameters - Day 92 Relative to Start Date

Sex: Female

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

[G]

Hb (mmol/L)

[G]

PCV (L/L)

[G]

MCV (fL)

[G]

MCH

(fmol)

 

[G]

MCHC

(mmol/L)

 

[G1]

Reticulo cytes

(%)

 [G]

Thrombo cytes

(10E9/L)

 [G]

Prothrom Time

(s)

[G1]

0 mg/kg

Mean SD

N

8.289

0.267

10

9.28

0.20

10

0.4672

0.0083

10

56.39

1.20

10

1.120

0.030

10

19.86

0.23

10

2.464

0.499

10

801.9

80.0

10

19.48

0.83

10

10 mg/kg

Mean SD

N

8.391

0.383

10

9.48

0.24

10

0.4754

0.0188

10

56.68

1.30

10

1.131

0.032

10

19.95

0.40

10

2.609

0.620

10

765.5

53.9

10

19.06

0.44

10

50 mg/kg

Mean

SD

N

8.352

0.240

10

9.45

0.32

10

0.4709

0.0130

10

56.40

1.26

10

1.132

0.039

10

20.07

0.40

10

2.570

0.288

10

831.7

102.1

10

19.42

1.24

10

250 mg/kg

Mean SD

N

8.489

0.401

10

9.29

0.33

10

0.4544

0.0186

10

53.56 ** 1.60

10

1.095

0.040

10

20.45 ** 0.25

10

2.902

0.572

10

922.2 * 121.8

10

18.50

0.65

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

 

Table 5. Total and differential white blood cell counts - Day 91 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G]

Eosino Absolute

(10E9/L)

 

[G1]

Baso Absolute

(10E9/L)

 

[G2]

Mono Absolute

(10E9/L)

 

[G]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G2]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

5.90

1.37

10

4.73

1.09

10

0.97

0.33

10

0.083

0.035

10

0.011

0.003

10

0.071

0.023

10

80.25

4.41

10

16.46

4.44

10

1.40

0.53

10

0.18

0.04

10

1.21

0.33

10

10 mg/kg

Mean SD

N

5.64

1.01

10

4.40

0.68

10

1.04

0.32

10

0.079

0.028

10

0.012

0.007

10

0.082

0.025

10

78.40

3.00

10

18.17

2.72

10

1.38

0.43

10

0.20

0.09

10

1.45

0.37

10

50 mg/kg

Mean

SD

N

5.53

1.05

10

4.27

0.84

10

1.09

0.30

10

0.066

0.018

10

0.010

0.004

10

0.084

0.032

10

77.21

4.28

10

19.58

4.13

10

1.20

0.27

10

0.18

0.08

10

1.50

0.43

10

250 mg/kg

Mean SD

N

5.80

1.93

10

4.35

1.56

10

1.26

0.48

10

0.068

0.042

10

0.008

0.007

10

0.082

0.032

10

74.76 * 5.09

10

22.05 * 5.22

10

1.18

0.66

10

0.13

0.09

10

1.44

0.37

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 6. Total and differential white blood cell counts – Day 92 Relative to Start Date

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G]

Eosino Absolute

(10E9/L)

 

[G1]

Baso Absolute

(10E9/L)

 

[G]

Mono Absolute

(10E9/L)

 

[G2]

Lympho cytes

(%)

 

[G1]

Neutro phils

(%)

 

[G1]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

4.60

1.21

10

3.72

1.04

10

0.67

0.17

10

0.083

0.049

10

0.011

0.005

10

0.085

0.033

10

80.77

3.05

10

14.95

3.34

10

1.70

0.59

10

0.24

0.11

10

1.88

0.70

10

10 mg/kg

Mean SD

N

4.95

1.41

10

3.97

1.19

10

0.78

0.22

10

0.074

0.074

10

0.013

0.006

10

0.094

0.037

10

79.66

4.11

10

16.27

4.42

10

1.47

1.11

10

0.26

0.10

10

1.88

0.55

10

50 mg/kg

Mean

SD

N

3.94

1.29

10

3.17

1.12

10

0.63

0.19

10

0.046

0.016

10

0.009

0.007

10

0.076

0.039

10

80.03

4.69

10

16.34

4.75

10

1.19

0.34

10

0.21

0.14

10

1.88

0.52

10

250 mg/kg

Mean SD

N

4.53

1.21

10

3.54

1.11

10

0.78

0.21

10

0.060

0.023

10

0.011

0.006

10

0.122

0.067

10

77.49

6.77

10

17.86

5.96

10

1.42

0.76

10

0.22

0.09

10

2.59

0.90

10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 7. Clinical chemistry - Day 91 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

[G]

ASAT (U/L)

[G1]

ALAT (U/L)

[G2]

GGT (U/L)

[G1]

Bilirub Total

(umol/L)

 

[G2]

Creatin ine

(umol/L)

 

[G2]

Total Protein

(g/L)

 

[G1]

Albumin (g/L)

[G2]

Albumin/ Globulin

 

[G2]

Glucose Plasma

(mmol/L)

 

[G2]

Cholest erol

(mmol/L)

 

[G2]

Triglyc erides

(mmol/L)

 

[G1]

Urea (mmol/L)

[G2]

PO4

 

(mmol/L)

 

[G2]

Ca (mmol/L)

[G2]

Cl (mmol/L)

[G1]

K

 

(mmol/L)

 

[G2]

Na (mmol/L)

[

G1]

T4

 

(ng/ml)

0 mg/kg

Mean SD

N

83.4

16.9

10

53.8

11.0

10

51.9

10.5

10

10.40

1.78

10

1.11

0.54

10

35.3

4.1

10

66.8

2.1

10

12.4

0.7

10

0.228

0.016

10

6.844

0.762

10

1.533

0.329

10

0.700

0.222

10

5.17

0.76

10

2.373

0.399

10

2.803

0.061

10

103.7

1.1

10

5.52

0.37

10

148.9

0.9

10

529.49

101.21

10

10 mg/kg

Mean SD

N

77.6

15.9

10

45.1

13.4

10

47.4

10.0

10

11.90

0.57

10

0.90

0.34

10

34.8

3.8

10

65.8

2.1

10

11.8

0.6

10

0.219

0.015

10

7.216

0.822

10

1.463

0.315

10

0.682

0.212

10

5.31

0.85

10

2.426

0.406

10

2.783

0.066

10

103.7

0.8

10

5.52

0.28

10

149.1

0.9

10

550.22

129.92

10

50 mg/kg

Mean

SD

N

87.0

20.0

10

49.3

19.1

10

41.3 *

9.2

10

11.00

1.49

10

1.07

0.68

10

33.4

4.2

10

63.6 ** 2.0

10

11.3 *

1.2

10

0.217

0.026

10

6.476

0.760

10

1.780

0.281

10

0.669

0.253

10

5.16

0.89

10

2.474

0.202

10

2.765

0.075

10

103.5

0.8

10

5.80

0.41

10

148.1

1.1

10

497.04

79.73

10

250 mg/kg

Mean SD

N

91.0

30.6

10

65.4 *

4.3

10

57.3

6.9

10

10.90

1.37

10

0.92

0.56

10

34.6

4.2

10

59.9 ** 1.6

10

10.0 **

0.7

10

0.201 ** 0.017

10

7.194

0.720

10

1.805

0.247

10

0.963

0.563

10

4.92

0.69

10

2.966 **

0.336

10

2.760

0.073

10

103.6

1.3

10

5.63

0.48

10

148.4

2.0

10

492.93

118.59

10

[G] - Ancova/Anova & Dunnett(Log)

[G1] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05; ** = p < 0.01

[G2] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

 

Table 8. Clinical chemistry - Day 92 Relative to Start Date

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

[G]

ASAT (U/L)

[G1]

ALAT (U/L)

[G2]

GGT (U/L)

[G2]

Bilirub Total

(umol/L)

 

[G2]

Creatin ine

(umol/L)

 

[G2]

Total Protein

(g/L)

 

[G2]

Albumin (g/L)

[G2]

Albumin/ Globulin

 

[G2]

Glucose Plasma

(mmol/L)

 

[G2]

Cholest erol

(mmol/L)

 

[G1]

Triglyc erides

(mmol/L)

 

[G]

Urea (mmol/L)

[G1]

PO4

 (mmol/L)

 

[G2]

Ca (mmol/L)

[G2]

Cl (mmol/L)

[G2]

K

 (mmol/L)

 

[G2]

Na (mmol/L)

[G2]

T4

 (ng/ml)

0 mg/kg

Mean SD

N

44.2

13.9

10

78.7

18.2

10

55.9

18.5

10

10.40

2.07

5

0.98

0.39

10

41.0

6.2

10

66.3

2.8

10

13.5

1.2

10

0.256

0.022

10

5.500

0.847

10

1.370

0.296

10

0.538

0.306

10

5.99

0.87

10

1.987

0.328

10

2.697

0.041

10

105.8

0.9

10

5.38

0.32

10

147.5

1.8

10

490.59

109.91

10

10 mg/kg

Mean SD

N

52.4

19.0

10

67.6

12.2

10

56.3

21.8

10

8.80

1.10

5

0.98

0.45

10

42.2

4.9

10

66.7

3.2

10

12.9

1.3

10

0.240

0.019

10

5.451

0.473

10

1.325

0.089

10

0.682

0.298

10

6.20

0.92

10

1.930

0.297

10

2.703

0.075

10

105.4

1.5

10

5.50

0.24

10

146.5

1.2

10

406.54

162.22

10

50 mg/kg

Mean

SD

N

45.1

7.6

10

65.5

19.8

10

38.2 *

9.2

10

10.86

0.90

7

1.03

0.52

10

37.3

5.2

10

64.2

1.9

10

12.8

0.9

10

0.249

0.020

10

5.843

0.627

10

1.614

0.391

10

0.729

0.346

10

6.79

0.94

10

2.117

0.314

10

2.712

0.071

10

105.5

1.4

10

5.50

0.44

10

146.5

1.8

10

370.76

175.21

10

250 mg/kg

Mean SD

N

47.7

20.6

10

60.9

16.9

10

37.3 *

7.5

10

11.00

1.58

5

0.83

0.48

10

36.4

2.9

10

64.0

3.2

10

11.4 **

1.3

10

0.216 ** 0.018

10

6.067

0.606

10

1.637

0.327

10

1.034

1.086

10

6.92

1.86

10

2.447 ** 0.263

10

2.739

0.105

10

103.9 ** 1.0

10

5.50

0.34

10

146.1

1.2

10

304.62

76.65

10

[G] - Ancova/Anova & Dunnett(Log)

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

 

Table 9. Relative organ weights - Day 91 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal

body wgt (g)

 

[G]

Brain

rel.wgt (g/kg body wgt)

[G1]

Heart

rel.wgt (g/kg body wgt)

[G]

Adrenals

rel.wgt (g/kg body wgt)

[G]

Kidneys

rel.wgt (g/kg body wgt)

[G1]

Liver

rel.wgt (g/kg body wgt)

[G]

Spleen

rel.wgt (g/kg body wgt)

[G]

Thymus

rel.wgt (g/kg body wgt)

[G]

Thyroid

rel.wgt (g/kg body wgt)

[G]

Testes

rel.wgt (g/kg body wgt)

[G]

Epididy

rel.wgt (g/kg body wgt)

[G]

Prostate

rel.wgt (g/kg body wgt)

[G]

Sem ves

rel.wgt (g/kg body wgt) [G]

0 mg/kg

Mean

SD

N

371.89

18.22

10

5.647

0.338

10

2.666

0.150

10

0.1393

0.0319

10

5.432

0.332

10

22.77

1.38

10

1.509

0.197

10

0.923

0.220

10

0.0404

0.0128

10

9.256

0.530

10

3.353

0.250

10

2.661

0.478

10

3.318

0.463

10

10 mg/kg

Mean

SD

N

358.31

27.22

10

5.749

0.440

10

2.662

0.216

10

0.1434

0.0162

10

5.357

0.163

10

22.38

0.69

10

1.498

0.182

10

1.033

0.189

10

0.0403

0.0108

9

9.581

0.847

10

3.395

0.336

10

2.678

0.460

10

2.900

0.425

10

50 mg/kg

Mean

SD

N

357.66

30.26

10

5.736

0.563

10

2.617

0.166

10

0.1465

0.0112

10

6.071 **

0.412

10

24.25 *

1.46

10

1.597

0.093

10

0.931

0.129

10

0.0428

0.0078

10

10.208 *

0.815

10

3.459

0.446

10

2.781

0.384

10

2.975

0.762

10

250 mg/kg

Mean

SD

N

331.98 **

23.83

10

5.929

0.391

10

2.678

0.139

10

0.1664 *

0.0229

10

6.922 **

0.546

10

28.27 **

1.21

10

1.687

0.203

10

0.757

0.196

10

0.0417

0.0117

10

10.581 **

1.040

10

3.666

0.377

10

2.969

0.587

10

3.011

0.655

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1] - Ancova/Anova & Dunnett(Log): ** = p < 0.01

 

Table 10. Relative organ weights - Day 92 Relative to Start Date

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G]

Spleen rel.wgt

(g/kg body wgt)

[G]

Thymus rel.wgt

(g/kg body wgt)

[G]

Thyroid rel.wgt

(g/kg body wgt)

[G]

Ovaries rel.wgt

(g/kg body wgt)

[G]

Uterus rel.wgt

(g/kg body wgt)

[G1]

0 mg/kg

Mean SD

N

207.72

15.06

10

8.914

0.616

10

2.996

0.172

10

0.2519

0.0225

10

6.008

0.403

10

23.52

1.53

10

1.794

0.135

10

1.461

0.232

10

0.0480

0.0114

10

0.3946

0.0811

10

3.483

1.450

10

10 mg/kg

Mean SD

N

209.21

16.39

10

8.985

0.641

10

2.994

0.210

10

0.2699

0.0497

10

6.032

0.505

10

24.19

2.05

10

2.018

0.274

10

1.396

0.168

10

0.0481

0.0105

10

0.4106

0.0947

10

3.749

1.950

10

50 mg/kg

Mean

SD

N

201.74

10.23

10

9.193

0.485

10

3.132

0.120

10

0.2847

0.0389

10

6.290

0.345

10

27.02 ** 2.20

10

1.968

0.225

10

1.360

0.205

10

0.0473

0.0179

10

0.4182

0.0730

10

3.780

1.701

10

250 mg/kg

Mean SD

N

196.54

17.38

10

9.158

0.769

10

3.044

0.243

10

0.2887

0.0572

10

6.794 **

0.383

10

30.08 ** 1.98

10

2.221 ** 0.217

10

1.037 ** 0.120

10

0.0448

0.0144

10

0.3449

0.0480

10

3.045

2.224

10

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

Table 11. Absolute organ weights – Day 91 relative to start date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal

body wgt (g)

 

[G]

Brain

 

(g)

 

[G1]

Heart

 

(g)

 

[G]

Adrenals

 

(g)

 

[G]

Kidneys

 

(g)

 

[G2]

Liver

 

(g)

 

[G]

Spleen

 

(g)

 

[G]

Thymus

 

(g)

 

[G]

Thyroid

 

(g)

 

[G2]

Testes

 

(g)

 

[G1]

Epididy

mides (g)

 

[G]

Prostate

 

(g)

 

[G]

Seminal

vesicles (g)

 

[G]

0 mg/kg

Mean

SD N

371.89

18.22

10

2.096

0.087

10

0.992

0.081

10

0.0515

0.0103

10

2.019

0.141

10

8.462

0.598

10

0.3424

0.0781

10

0.0150

0.0049

10

3.442

0.259

10

1.246

0.100

10

0.988

0.176

10

1.231

0.154

10

0.5594

0.0591

10

10 mg/kg

Mean

SD

N

358.31

27.22

10

2.050

0.064

10

0.950

0.059

10

0.0512

0.0057

10

1.918

0.138

10

8.019

0.681

10

0.3688

0.0656

10

0.0142

0.0036

9

3.419

0.217

10

1.212

0.104

10

0.961

0.182

10

1.036

0.153

10

0.5367

0.0771

10

50 mg/kg

Mean

SD

N

357.66

30.26

10

2.037

0.059

10

0.933

0.060

10

0.0524

0.0060

10

2.169

0.215

10

8.690

1.061

10

0.3311

0.0400

10

0.0152

0.0025

10

3.637

0.260

10

1.231

0.138

10

0.996

0.175

10

1.064

0.279

10

0.5707

0.0553

10

250 mg/kg

Mean

SD

N

331.98 **

23.83

10

1.960 **

0.032

10

0.890 *

0.089

10

0.0550

0.0070

10

2.303 *

0.295

10

9.386 *

0.778

10

0.2510 **

0.0652

10

0.0139

0.0043

10

3.500

0.281

10

1.216

0.146

10

0.989

0.220

10

1.004

0.256

10

0.5583

0.0604

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

[G2] - Ancova/Anova & Dunnett(Log): * = p < 0.05

 

Table 12. Absolute oran weights – Day 92 relative to start date

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain (g)

[G]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G]

Spleen (g)

[G]

Thymus (g)

[G]

Thyroid (g)

[G]

Ovaries (g)

[G]

Uterus (g)

[G1]

0 mg/kg

Mean SD

N

207.72

15.06

10

1.844

0.051

10

0.622

0.052

10

0.0522

0.0044

10

1.249

0.134

10

4.887

0.486

10

0.3733

0.0466

10

0.3032

0.0512

10

0.0100

0.0027

10

0.0821

0.0183

10

0.7240

0.3102

10

10 mg/kg

Mean SD

N

209.21

16.39

10

1.871

0.057

10

0.625

0.048

10

0.0564

0.0113

10

1.259

0.111

10

5.050

0.463

10

0.4202

0.0511

10

0.2920

0.0402

10

0.0101

0.0026

10

0.0850

0.0169

10

0.7942

0.4500

10

50 mg/kg

Mean

SD

N

201.74

10.23

10

1.851

0.056

10

0.632

0.043

10

0.0576

0.0094

10

1.267

0.058

10

5.448 * 0.476

10

0.3973

0.0524

10

0.2740

0.0424

10

0.0095

0.0035

10

0.0843

0.0149

10

0.7547

0.3218

10

250 mg/kg

Mean SD

N

196.54

17.38

10

1.788

0.040

10

0.597

0.060

10

0.0562

0.0091

10

1.335

0.138

10

5.907 **

0.593

10

0.4349 *

0.0424

10

0.2044 **

0.0356

10

0.0087

0.0024

10

0.0677

0.0108

10

0.6181

0.4916

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

Table 13. Percentage change in relative organ weights compared to controls – Day 91 and 92 Relative to Start Date for males and females, respectively.

Group

Relative Liver weight

Relative Kidney weight

Relative Spleen weight

Relative Thymus weight

Relative adrenal weight

males

female s

males

females

males

females

males

females

males

females

Low-dose

- 2%

+ 3%

- 1%

0%

- 1%

+12%

+12%

- 4%

+ 3%

+ 7%

Mid-dose

+ 6%*

+15%**

+12%**

+ 5%

+ 6%

+10%

+ 1%

- 7%

+ 5%

+13%

High-dose

+24%**

+28%**

+27%**

+13%**

+12%

+24%**

-18%

-29%**

+19%*

+15%

  * Statistically significant (p < 0.05); ** Statistically significant (p < 0.01).

 

 Table 14. Historical TSH in serum data

 

Males

Females

Study type

OECD 443

OECD 443

OECD 4081

OECD 408

OECD 443

OECD 443

OECD 4081

OECD 408

Method

Elisa

Elisa

Elisa

Elisa

Elisa

Elisa

Elisa

Elisa

Medium

serum

Serum

serum

serum

serum

serum

serum

serum

Approx. age of rats

22 weeks

13 weeks

19 weeks

19 weeks

19 weeks

13 weeks

19 weeks

19 weeks

Mean

2661

3872

2969

2419

1923

983

3683

2978

SD

1174.6

1047.5

798.6

728.8

692.0

567.6

1134.2

551.6

CV

44.1%

27.1%

26.9 %

30.1%

36.0%

57.7%

30.8 %

18.5%

Count

10

8

10

10

10

9

10

10

¹ Current study

 Table 15. Historical T4 data

 

Males

Females

Study type

OECD 443

OECD 443

OECD 4081

OECD 4081

OECD 408

OECD 443

OECD 443

OECD 4081

OECD 4081

OECD 408

Method

Elisa²

Elisa²

Elisa²

Siemens³

Siemens³

Elisa²

Elisa²

Elisa²

Siemens³

Siemens³

Medium

serum

serum

serum

plasma

plasma

serum

serum

serum

plasma

plasma

Approx. age of rats

22 weeks

13 weeks

19 weeks

19 weeks

19 weeks

19 weeks

13 weeks

19 weeks

19 weeks

19 weeks

Mean

81.8

197.5

400.8

529.5

532.4

69.0

60.6

418.4

490.6

468.3

SD

26.6

45.6

131.8

101.2

103.4

22.0

15.6

66.2

109.9

93.4

CV

32.6%

23.1%

32.9%

 

 

31.8%

25.7%

15.8%

 

 

Count

10

8

10

10

10

10

9

10

10

10

¹ Current study

² The numeral values obtained may differ with various commercial Elisa assay kits. Therefore, only one test kit and lot number was used within one study.

³ Siemens Dimension ExL, Clinical Chemistry system

Table 16. Hormone determinations in males and females – Day 91 and 92 relative to start date, respectively.

 

Male

Male

Male

Female

Female

Female

TSH

(pg/ml) [G]

T4

(ng/ml) [G1]

(serum)

T4

(ng/ml) [G]

(plasma)

TSH

(pg/ml) [G1]

T4

(ng/ml) [G2]

(serum)

T4

(ng/ml) [G2]

(plasma)

0 mg/kg

Mean

2969.06

400.77

529.46

3682.50

418.36

490.59

 

SD

798.56

131.79

101.21

1134.23

66.18

109.91

 

N

10

10

10

10

10

10

10 mg/kg

Mean

2484.13

316.79

550.22

4758.34

438.78

406.54

 

SD

714.98

90.88

129.92

2081.07

145.54

162.22

 

N

10

10

10

10

10

10

50 mg/kg

Mean

3530.56

639.94

497.04

4063.44

622.20

370.76

 

SD

955.78

384.86

79.73

955.04

307.14

175.21

 

N

10

10

10

10

10

10

250 mg/kg

Mean

2623.23

495.51

492.93

3349.18

469.32

304.62**

 

SD

775.32

191.28

118.59

774.96

109.20

75.65

 

N

10

10

10

10

10

10

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks: ** = p<0.01

Conclusions:
In this GLP complaint oral repeated dose toxicity study, performed according to OECD 408, the no-observed-adverse-effect level (NOAEL) is conservatively placed at the lowest level tested (10 mg/kg body weight/day) based on increased relative liver (both sexes) and kidney (males) weight in combination with effects on total protein and albumin in the mid-dose group (males).
Based on this OECD 408 study and preliminary data from an OECD 414 study, a need for changes to the study design of the OECD 443 study is identified. Considerations for this change can be found in section 7.8.1 of this dossier under ‘Toxicity to reproduction – waiver/change in study design’ in the 'Justification for type of information' field.
Executive summary:

In a GLP compliant study, performed according to OECD 408, the safety of the test substance ZDBC was examined in a sub-chronic (13 week) oral toxicity study in Wistar rats. ZDBC was administered by daily oral gavage as a suspension in corn oil at levels of 0 (vehicle control), 10, 50 and 250 mg/kg body weight/day to groups of 10 rats/sex during 13 weeks. Clinical signs, body weight, food and water consumption were monitored throughout the study. Ophthalmoscopic examinations were performed before dosing commenced for all animals and towards the end of treatment in the control and high-dose group. Behavioural endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats at the end of the study (in week 12). Haematology, clinical chemistry and urinalysis were performed at the end of the study. All animals were killed, subjected to necropsy and post mortem examination, major organs were weighed and a full range of tissues were examined microscopically.

There was no mortality. Clinical observations noted in the high-dose group included sliding with the ventral parts of the head and neck over the bottom of the open field, piloerection, salivation, occasional hypoactivity, and, in males only, decreased hindlimb gripstrength and increased body temperature. In the mid-dose group, piloerection and, in males only, salivation and a decrease in hindlimb gripstrength were noted. Ophthalmoscopy was not affected and there were no indications of any neurotoxic potential of ZDBC. Body weights were slightly (≤9%) reduced in high-dose males. There were no relevant changes in food or water intake.Haematology was conducted in 10 rats/sex/group at necropsy.Thrombocytes were increased in high-dose females. There were no other relevant changes in red blood cell parameters or in total or differential white blood cell counts.Clinical chemistry, conducted in 10 rats/sex/group at necropsy, showeda dose-related decrease in thetotal protein and albumin concentrations in males of the mid- and high-dose groups, while the A/G ratio was decreased in high-dose males. In high-dose females, albumin concentration and A/G ratio were decreased.Furthermore, inorganic Phosphate concentration was increased in the high-dose group in both sexes. Thyroid Stimulating Hormone (TSH) and Thyroxine (T4) levels, as assessed in serum using ELISA, were not affected by the treatment. T4 hormone levels in plasma (inadvertently measured by the Siemens Dimension Clinical Chemistry system) were, however, found to be lower in high-dose females compared to concurrent controls. This intergroup difference in plasma T4 levels in high-dose females probably reflects normal background variation. Unfortunately it is difficult to provide performance criteria for T4, because our historical control data base is rather limited, especially for T4 analysis in plasma by Siemens Dimension Clinical Chemistry system (for which only one other study is currently available). The lower plasma T4 was not corroborated by treatment-related changes in TSH levels, thyroid weight and pathology, or by effects on plasma total cholesterol levels which are known to be controlled by thyroid hormone action (OECD Guideline 408, 2018). Moreover, this finding was not supported by similar results on T4 obtained by Elisa. Therefore the lower T4 in plasma of high-dose females as obtained by the Siemens Dimension Clinical Chemistry system was considered to be a chance finding. In summary, the weight of evidence from this study does not indicate a perturbation of the thyroid pathway. Urinalysis was conducted in 10 rats/sex/group in week 13 of the study.The urinary volume was increased in high-dose males. Dipstick measurements showed an increase in urinary ketones in high-dose females, mainly due to an increased severity in three of these females. For the high-dose group, the following changes in organ weights were ascribed to treatment: increased relative weights of liver and kidneys (both sexes), the spleen (females) and adrenals (males) and decreased thymus weight (both sexes). Some of these changes were still observed in the mid-dose group: increased relative weights of the liver (both sexes) and kidneys (males). Macroscopic examination at necropsy and microscopic examination of organs and tissues did not reveal treatment-related findings.

Although microscopic examination of organs and tissues did not reveal treatment-related findings in any group, the increased liver and kidney weight in combination with effects on total protein and albumin in the mid-dose group cannot completely be disregarded. Therefore, the no-observed-adverse-effect level (NOAEL) is conservatively placed at the lowest level tested (10 mg/kg body weight/day).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant, OECD 408
System:
hepatobiliary
Organ:
liver

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

Oral

Triskelion B.V. 2018

In a GLP compliant study, performed according to OECD 408, the safety of the test substance ZDBC was examined in a sub-chronic (13 week) oral toxicity study in Wistar rats. ZDBC was administered by daily oral gavage as a suspension in corn oil at levels of 0 (vehicle control), 10, 50 and 250 mg/kg body weight/day to groups of 10 rats/sex during 13 weeks.

Treatment-related effects noted in the high-dose group included clinical observations, slight reduction in male body weights, increased thrombocytes (females), decreased total protein and/or albumin concentrations and A/G ratio (both sexes), increased inorganic phosphate concentration (both sexes), increased urinary volume (males) and ketones (females), increased relative weights of liver and kidneys (both sexes), the spleen (females) and adrenals (males) and decreased thymus weight (both sexes). Some of these changes were still observed in the mid-dose group, namely some clinical observations (piloerection, salivation and a decrease in male hindlimb grip strength), decreased total protein and albumin concentrations in males and increased relative weights of the liver (both sexes) and kidneys (males).

Gray 1977

One oral repeated dose toxicity study with Wistar rats was available for assessment (Gray et al., 1977). Groups of 15 male and 15 female rats were given diets containing 9, 100, 500 2500 ppm of zinc bis(dibutyldithiocarbamate) for 17 weeks. Additional groups of 5 rats of each sex and of similar body weight were given diets containing 0, 500 or 2500 ppm zinc dibutyldithiocarbamate for 2 or 6 weeks. At the end of the appropriate period of feeding the rats were killed and necropsied. In addition to gross pathology, histopathological examinations, haematological and clinical chemistry examinations were performed.

At the highest treatment level the rate of body weight gain of the females and the food intake of both sexes were reduced. The reduction in food intake was maximal during the first few days of the study, a pattern often associated with an unpalatable diet. There were no effects attributable to the test substance in the results of haematological examination or the analyses of serum and urine. The relative weights of the kidneys and liver were significantly increased in rats of both sexes given 2500 ppm zinc bis(dibutyldithiocarbamate) for 17 weeks. No histopathological changes were however observed in these organs, and, in case of kidneys, the renal function tests provided no evidence of any functional impairment. An increase in liver weight unaccompanied by histological changes, when accompanied by increased levels of the hepatic microsomal drug-metabolising enzymes, is usually considered an adaptive physiological response to increased metabolic demand. However, in the present case, due to the absence of additional biochemical data, it is not possible to determine whether the observed increase in the relative liver weight due to the treatment with zinc bis(dibutyldithiocarbamate) can be explained in this way.

Based on the increased relative liver and kidney weights at the highest dietary level, the NOAEL was set at 500 ppm in diet, corresponding to overall intakes of the test substance of 41 and 47 mg/kg bw/day for male and female rats, respectively.

Inhalation and dermal

No data on repeated dose toxicity of zinc bis(dibutyldithiocarbamate) (ZDBC) by administration via inhalation or dermal route are available.

Justification for classification or non-classification

Based on the NOAEL of 10 mg/kg bw/day obtained in the 13-weeks repeated dose toxicity study with rats, which is below the cut-off value of 50 mg/kg bw/day established by EU Directive 67/548/EEC and the cut-off value of 100 mg/kg bw/day established by EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 a classification of zinc bis(dibutyldithiocarbamate) (ZDBC) as harmful: danger of serious damage to health by prolonged exposure via oral route (Xn, R48/20) could be considered according to EU Directive 67/548/EEC. However, according to these Directive and Regulation, classification should not be applied when the effects are limited to changes in organ weights with no evidence of organ dysfunction. As the increases in organ weights were not accompanied by histopathological changes, and, in case of kidneys, no functional impairment was observed, classification of zinc bis(dibutyldithiocarbamate) is not warranted according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.