Zinc bis(dibutyldithiocarbamate)

Endpoint summary

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

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 21st September 1998.

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

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 or test system 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 408^1	OECD 408	OECD 443	OECD 443	OECD 408^1	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 408^1	OECD 408^1	OECD 408	OECD 443	OECD 443	OECD 408^1	OECD 408^1	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.