Zinc bis(dibenzyldithiocarbamate)

Endpoint summary

Description of key information

- In a GLP complaint oral repeated dose toxicity study, performed according to OECD 408, the no-observed-adverse-effect level (NOAEL) is placed at the highest level tested (1000 mg/kg body weight/day) because no adverse effects were observed.

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:

5 Apr 2018 to 6 Jul 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: 6 to 7 weeks.
- Weight at study initiation: For males: 146 to 173g (mean 160 g); for females: 114 to 154 g (mean 133 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: 21 March to 23 March. Further acclimation until initiation of treatment on 5th of April.

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:
5 Apr 2018 to 6 Jul 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
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 8, 40 and 200 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 5 April 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 5 April, 30 April, 28 May and 27 June 2018.

Duration of treatment / exposure:

13 consecutive weeks.

Frequency of treatment:

Daily, seven days a week.

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

Group 2, low-dose.

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Group 3, mid-dose.

Dose / conc.:

1 000 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

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, rats were 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 -1, 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 -8 (all males), day -7 (all females), and in all rats of the control group and the high-dose group in week 13 (day 88).

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: In or around week 13.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for 16 hours.
- 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 for males and week 13 for females).

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.
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

Sacrifice and pathology:

SACRIFICE:
Early in week 14, after overnight fasting (water was freely available), the surviving animals were killed on two successive working days (day 91 and 92 for males and females, respectively), in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes.

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 (brain stem, cerebrum, cerebellum), 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 (cervical, mid-thoracic, lumbar), femur with joint*, spleen, GALT (gut associated lymphoid tissue, including Peyer's patches), sternum with bone marrow, stomach ('forestomach', fundus, pylorus), 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.

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:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs

Mortality:

no mortality observed

Description (incidence):

None of the rats died during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the final stage of the study, mean body weights tended to be slightly (≤8%) higher in males of the mid- and high-dose groups, but there were no statistically significant differences between the treatment groups and the controls (Table 1 and 2 in 'Any other information on results incl. tables').
Incidental changes in body weight gain attained statistical significance in females of the low and mid-dose group in the pre-dose phase, and in males of the mid-dose group in week 5 (Table 3 and 4 in 'Any other information on results incl. tables'). The overall growth figures were, however, not adversely affected.
In conclusion; body weights and body weight gain were not adversely affected by the test substance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intake was not affected by the treatment. Food intake was slightly though statistically significantly decreased in males in the low-dose groups in week 4. There were, however, no noticeable differences in overall food intake among the groups.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Water intake was not affected by the treatment. On day 70-71, water intake was statistically significantly lower in female rats in the high dose groups than in controls, but there were no noticeable differences in overall weekly water consumption among the groups in the various weeks.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of all animals in the control and high-dose group in week 13 did not reveal any changes.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

An overview of the results can be found in Table 5 to 8 in 'Any other information on results incl. tables'.
Red blood cell count, haemoglobin concentration and packed cell volume were statistically significantly decreased in females of the high dose group. Although the changes in this group were outside the range of historical control data, they were only slight (about -6%).
A slight, though statistically significant increase in the percentage of reticulocytes in low-dose males was not confirmed at the higher dose levels and is therefore considered an incidental finding.
The percentage of lymphocytes was statistically significantly increased and the percentage of neutrophils was decreased in males of the high-dose group. These findings were within the range of historical control data, and not reflected in significant changes in the absolute lymphocyte and neutrophil counts. Therefore these findings are of doubtful relevance and are not considered to be adverse.
An increase in the absolute number of neutrophils in mid-dose females was not confirmed at the high-dose level and therefore considered an incidental finding.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An overview of the results can be found in Table 9 and 10 in 'Any other information on results incl. tables'.
Albumin and the albumin/globulin ratio were statistically significantly decreased in males of the mid- and high-dose group. The changes were within the range of historical control data.
Chloride concentration was statistically significantly decreased in high-dose females. The changes were within the range of historical control data.
A number of changes attained statistical significance but were not confirmed at higher dose level(s), and are therefore considered chance findings:
- Decrease in ALP activity in mid-dose males.
- Decreases in potassium concentration in low- and mid-dose males.
- Decrease in total protein in low-dose females.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in urinary volume or density, or in semi-quantitative (dipstick) urinary measurements.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute weight of the liver was statistically significantly increased in male animals of the mid- and high-dose group (Table 11 and 12 in 'Any other information on results incl. tables'). The relative weight of this organ was not affected (Table 13 and 14 in 'Any other information on results incl. tables'). Therefore this finding is ascribed to the somewhat higher terminal body weights in these groups and not considered to be of toxicological significance.
A statistically significant decrease in the relative weight of the heart in mid-dose females was considered a chance finding.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONE LEVELS
No statistically significant effects in plasma T4 levels were noted between treatment groups and controls (Table 15 and 16 in 'Any other information on results incl. tables').
Thyroid stimulating hormone levels were slightly decreased in females of the high-dose group. This decrease in TSH levels was not corroborated by noticeable changes in T4 levels, thyroid weight or pathology, or by effects on plasma total cholesterol. Since, moreover the values were well within the range of historical control data and this variable is known to show considerable variation, the decrease in TSH levels in high-dose females was considered a chance finding (Table 15 and 16 in 'Any other information on results incl. tables').

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Critical effects observed:

no

Table 1. Body weights males – days relative to start date

Sex: Male		Bodywt day -x (g) [G]	Bodywt (g)   [G]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]	Bodywt (g)   [G1]
		-1	0	7	14	21	28	35	42	49	56	63	70	77	84	90
0 mg/kg	Mean SD N	156.01 8.84 10	159.82 8.60 10	197.22 7.39 10	231.42 7.03 10	256.04 9.75 10	275.02 15.25 10	288.00 17.15 10	298.59 17.36 10	308.30 18.90 10	315.75 19.25 10	323.28 21.05 10	331.75 21.69 10	338.57 21.93 10	334.59 21.47 10	349.08 21.20 10
40 mg/kg	Mean SD N	155.79 9.20 10	160.03 8.38 10	197.35 10.62 10	228.61 14.66 10	253.51 18.79 10	268.33 24.09 10	281.39 27.80 10	293.11 28.87 10	304.26 29.72 10	312.59 29.99 10	320.69 31.39 10	329.98 32.59 10	337.57 32.78 10	332.47 32.33 10	349.96 33.14 10
200 mg/kg	Mean SD N	155.70 9.43 10	160.13 9.00 10	200.23 9.11 10	235.66 9.37 10	264.63 12.54 10	285.48 16.67 10	305.17 20.48 10	319.88 23.04 10	330.93 23.79 10	340.05 26.39 10	346.83 29.44 10	357.75 31.01 10	363.36 31.53 10	357.73 30.07 10	373.58 33.31 10
1000 mg/kg	Mean SD N	155.94 9.36 10	160.21 9.47 10	199.91 11.62 10	233.85 13.11 10	263.57 14.20 10	283.98 17.55 10	301.84 17.61 10	315.39 18.95 10	328.75 19.36 10	337.88 19.34 10	348.12 20.65 10	358.94 20.28 10	365.88 19.45 10	361.50 21.33 10	376.86 22.16 10

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett

 

Table 2. Body weights females – days relative to start date

Sex: Female		Bodywt day -x (g)   [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G]	Bodywt (g) [G1]	Bodywt (g) [G]	Bodywt (g) [G]
		-1	0	7	14	21	28	35	42	49	56	63	70	77	84	90
0 mg/kg	Mean SD N	129.72 8.32 10	133.67 8.61 10	151.92 7.01 10	167.35 9.44 10	176.66 10.75 10	189.11 13.48 10	198.34 14.12 10	201.25 13.03 10	206.38 12.38 10	215.16 14.44 10	217.58 12.18 10	218.22 13.99 10	219.36 13.43 10	223.60 12.23 10	223.85 15.28 10
40 mg/kg	Mean SD N	130.73 9.57 10	132.71 9.21 10	151.78 10.39 10	168.01 11.74 10	180.11 13.69 10	190.67 15.94 10	198.38 14.80 10	202.20 15.71 10	205.08 15.01 10	211.81 15.11 10	214.01 16.10 10	216.43 17.00 10	216.44 15.43 10	217.46 15.76 10	222.42 17.07 10
200 mg/kg	Mean SD N	131.44 10.64 10	132.47 12.57 10	152.11 11.62 10	168.43 12.61 10	181.47 11.41 10	192.00 16.47 10	203.13 16.08 10	208.81 15.77 10	213.06 14.26 10	218.77 16.92 10	221.63 15.02 10	223.10 14.32 10	224.65 13.10 10	227.78 18.00 10	230.86 14.93 10
1000 mg/kg	Mean SD N	127.84 9.85 10	131.18 8.44 10	149.11 10.00 10	163.33 9.19 10	176.02 10.22 10	184.29 11.18 10	195.25 10.85 10	199.77 10.03 10	206.95 12.67 10	209.55 11.79 10	212.01 11.93 10	215.15 10.94 10	216.70 12.77 10	218.94 12.43 10	220.09 12.21 10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

 

Table 3. Body weight change males – days relative to the start date.

Sex: Male		Wgt change last -x to 0 (g) [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G1]	Body wt change (g)     [G2]	Body wt change (g)     [G1]
		-x - 0	0 - 7	7 - 14	14 - 21	21 - 28	28 - 35	35 - 42	42 - 49	49 - 56	56 - 63	63 - 70	70 - 77	77 - 84	84 - 90	0 - 90
0 mg/kg	Mean SD N	3.81 1.60 10	37.40 3.75 10	34.20 4.45 10	24.62 5.24 10	18.98 6.83 10	12.98 6.42 10	10.59 3.78 10	9.71 2.72 10	7.45 2.81 10	7.53 2.87 10	8.47 3.43 10	6.82 1.77 10	-3.98 2.84 10	14.49 1.98 10	189.26 25.54 10
40 mg/kg	Mean SD N	4.24 2.84 10	37.32 3.59 10	31.26 6.79 10	24.90 5.85 10	14.82 6.69 10	13.06 5.92 10	11.72 3.51 10	11.15 3.90 10	8.33 2.28 10	8.10 2.66 10	9.29 2.02 10	7.59 2.08 10	-5.10 4.84 10	17.49 4.57 10	189.93 28.07 10
200 mg/kg	Mean SD N	4.43 1.52 10	40.10 3.11 10	35.43 3.75 10	28.97 5.09 10	20.85 5.86 10	19.69 * 4.64 10	14.71 4.58 10	11.05 2.73 10	9.12 3.42 10	6.78 3.47 10	10.92 3.57 10	5.61 1.63 10	-5.63 4.12 10	15.85 4.84 10	213.45 32.73 10
1000 mg/kg	Mean SD N	4.27 1.32 10	39.70 3.50 10	33.94 3.84 10	29.72 6.24 10	20.41 4.67 10	17.86 4.67 10	13.55 3.34 10	13.36 2.89 10	9.13 2.58 10	10.24 3.64 10	10.82 3.31 10	6.94 2.56 10	-4.38 3.28 10	15.36 3.18 10	216.65 21.43 10

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

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

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 4. Body weight change females – days relative to the start date.

Sex: Female		Wgt change last -x to 0 (g) [G]	Body wt change (g)    [G1]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G2]	Body wt change (g)    [G]	Body wt change (g)    [G2]
		-x - 0	0 - 7	7 - 14	14 - 21	21 - 28	28 - 35	35 - 42	42 - 49	49 - 56	56 - 63	63 - 70	70 - 77	77 - 84	84 - 90	0 - 90
0 mg/kg	Mean SD N	3.95 2.79 10	18.25 3.52 10	15.43 4.94 10	9.31 9.35 10	12.45 4.77 10	9.23 3.43 10	2.91 3.91 10	5.13 7.26 10	8.78 6.57 10	2.42 3.72 10	0.64 3.62 10	1.14 6.99 10	4.24 4.46 10	0.25 5.97 10	90.18 14.98 10
40 mg/kg	Mean SD N	1.98 * 1.87 10	19.07 2.47 10	16.23 3.65 10	12.10 6.18 10	10.56 7.46 10	7.71 2.95 10	3.82 3.95 10	2.88 5.84 10	6.73 7.53 10	2.20 4.66 10	2.42 3.12 10	0.01 5.03 10	1.02 5.27 10	4.96 6.48 10	89.71 12.04 10
200 mg/kg	Mean SD N	1.03 ** 2.94 10	19.64 3.16 10	16.32 2.50 10	13.04 6.60 10	10.53 6.16 10	11.13 4.79 10	5.68 5.37 10	4.25 7.37 10	5.71 6.59 10	2.86 3.87 10	1.47 3.97 10	1.55 8.17 10	3.13 5.99 10	3.08 7.24 10	98.39 8.53 10
1000 mg/kg	Mean SD N	3.34 3.09 10	17.93 3.67 10	14.22 5.29 10	12.69 5.50 10	8.27 4.48 10	10.96 4.18 10	4.52 5.33 10	7.18 5.64 10	2.60 6.07 10	2.46 4.87 10	3.14 4.99 10	1.55 5.17 10	2.24 5.99 10	1.15 6.40 10	88.91 8.53 10

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

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

[G2] - Ancova/Anova & Dunnett

 

Table 5. Red blood cell and coagulation parameters males – day 91 relative to start date.

Sex: Male		RBC (10E12 /L) [G]	Hb (mmol /L) [G]	PCV (L/L)   [G]	MCV (fL)     [G]	MCH (fmol)   [G]	MCHC (mmol /L) [G]	Reticulo cytes (%) [G]	Thrombo cytes (10E9/L) [G]	Prothrom Time (s) [G]
0 mg/kg	Mean SD N	8.885 0.311 10	9.61 0.26 10	0.4748 0.0140 10	53.45 0.61 10	1.082 0.024 10	20.24 0.31 10	1.811 0.223 10	699.0 65.4 10	19.33 0.49 10
40 mg/kg	Mean SD N	8.916 0.315 10	9.59 0.31 10	0.4793 0.0149 10	53.77 1.03 10	1.076 0.024 10	20.01 0.39 10	2.178 ** 0.178 10	767.2 112.5 10	19.71 0.80 10
200 mg/kg	Mean SD N	8.879 0.324 10	9.52 0.26 10	0.4722 0.0162 10	53.20 1.17 10	1.073 0.018 10	20.17 0.31 10	1.989 0.233 10	748.4 88.2 10	19.57 0.65 10
1000 mg/kg	Mean SD N	8.967 0.403 10	9.64 0.31 10	0.4800 0.0170 10	53.56 1.26 10	1.076 0.026 10	20.09 0.35 10	1.862 0.194 10	732.0 86.8 10	19.56 0.89 10

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

 

Table 6. Red blood cell and coagulation parameters females – 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 (%) [G1]	Thrombo cytes (10E9/L) [G]	Prothrom Time (s) [G1]
0 mg/kg	Mean SD N	8.483 0.467 10	9.57 0.46 10	0.4691 0.0266 10	55.30 0.99 10	1.129 0.018 10	20.41 0.27 10	2.419 0.369 10	738.0 53.6 10	19.39 1.03 10
40 mg/kg	Mean SD N	8.320 0.339 10	9.21 0.28 10	0.4505 0.0137 10	54.17 0.84 10	1.107 0.020 10	20.44 0.17 10	2.504 0.463 10	749.3 92.9 10	19.71 0.60 10
200 mg/kg	Mean SD N	8.383 0.386 10	9.31 0.28 10	0.4556 0.0168 10	54.38 1.42 10	1.112 0.040 10	20.44 0.50 10	2.531 0.400 10	718.4 58.8 10	19.43 0.69 10
1000 mg/kg	Mean SD N	7.940 ** 0.341 10	8.98 ** 0.31 10	0.4391 ** 0.0144 10	55.34 1.61 10	1.132 0.039 10	20.45 0.32 10	2.534 0.176 10	716.0 73.6 10	19.60 0.33 10

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

[G1] - Kruskal-Wallis & Dunnett on Ranks

 

Table 7. Total and differential white blood cell counts males – 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) [G]	Baso Absolute (10E9/L) [G]	Mono Absolute (10E9/L) [G1]	Lympho cytes (%)   [G]	Neutro phils (%)   [G]	Eosino phils (%)   [G]	Baso phils (%)   [G]	Mono cytes (%)   [G1]
0 mg/kg	Mean SD N	6.16 1.00 10	4.76 0.87 10	1.14 0.20 10	0.125 0.046 10	0.015 0.007 10	0.095 0.028 10	77.00 2.89 10	18.74 3.46 10	2.02 0.63 10	0.24 0.12 10	1.53 0.32 10
40 mg/kg	Mean SD N	5.72 1.76 10	4.25 1.37 10	1.22 0.38 10	0.105 0.045 10	0.011 0.007 10	0.115 0.075 10	73.87 4.78 10	21.77 4.42 10	1.90 0.71 10	0.18 0.08 10	1.88 0.74 10
200 mg/kg	Mean SD N	6.32 1.74 10	5.01 1.46 10	1.06 0.30 10	0.105 0.038 10	0.014 0.007 10	0.100 0.038 10	79.02 3.59 10	16.97 2.93 10	1.73 0.83 10	0.21 0.07 10	1.56 0.39 10
1000 mg/kg	Mean SD N	6.35 1.21 10	5.19 1.09 10	0.92 0.25 10	0.097 0.031 10	0.014 0.006 10	0.097 0.031 10	81.56 * 3.81 10	14.74 * 3.39 10	1.55 0.46 10	0.21 0.07 10	1.52 0.34 10

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

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

 

Table 8. Total and differential white blood cell counts females – 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) [G1]	Mono Absolute (10E9/L) [G2]	Lympho cytes (%)   [G]	Neutro phils (%)   [G]	Eosino phils (%)   [G1]	Baso phils (%)   [G2]	Mono cytes (%)   [G]
0 mg/kg	Mean SD N	4.60 1.45 10	3.79 1.16 10	0.62 0.30 10	0.083 0.072 10	0.014 0.010 10	0.076 0.054 10	82.89 5.83 10	13.05 4.39 10	1.91 1.96 10	0.29 0.14 10	1.53 0.75 10
40 mg/kg	Mean SD N	4.56 1.26 10	3.62 1.12 10	0.74 0.20 10	0.086 0.028 10	0.012 0.009 10	0.087 0.045 10	78.57 5.88 10	16.94 5.79 10	1.96 0.61 10	0.26 0.18 10	1.89 0.64 10
200 mg/kg	Mean SD N	5.39 1.16 10	4.16 1.07 10	1.02 * 0.34 10	0.075 0.030 10	0.013 0.006 10	0.097 0.040 10	76.74 6.24 10	19.31 6.17 10	1.38 0.46 10	0.23 0.08 10	1.77 0.48 10
1000 mg/kg	Mean SD N	4.91 1.03 10	3.85 0.85 10	0.86 0.31 10	0.086 0.041 10	0.012 0.006 10	0.083 0.027 10	78.53 5.62 10	17.48 5.14 10	1.74 0.67 10	0.25 0.12 10	1.69 0.38 10

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

[G1] - Kruskal-Wallis & Dunnett on Ranks

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

 

Table 9. Clinical chemistry males – day 91 relative to start date.

Sex: Male		ALP (U/L) [G]	ASAT (U/L) [G]	ALAT (U/L) [G1]	GGT (U/L) [G]	Bilirub Total (umol/L) [G]	Creatin ine (umol/L) [G1]	Total Protein (g/L) [G2]	Albumin (g/L) [G1]	Albumin/ Globulin [G2]	Glucose Plasma (mmol/L) [G]	Cholest erol (mmol/L) [G]	Triglyc erides (mmol/L) [G1]	Urea (mmol/L) [G]	PO4 (mmol/L) [G]	Ca (mmol/L) [G]	Cl  (mmol/L) [G]	K (mmol/L) [G]	Na (mmol/L) [G]	T4 (ng/ml)
0 mg/kg	Mean SD N	99.9 23.3 10	65.8 10.6 10	53.5 14.3 10	6.80 1.03 10	1.04 0.31 10	34.5 5.5 10	64.3 2.3 10	12.6 1.3 10	0.244 0.029 10	6.750 0.827 10	1.826 0.192 10	0.762 0.199 10	5.32 0.55 10	2.380 0.387 10	2.727 0.064 10	105.2 0.8 10	5.79 0.38 10	149.1 1.2 10	532.37 103.37 10
40 mg/kg	Mean SD N	95.4 16.0 10	61.8 18.7 10	59.7 25.6 10	6.90 0.99 10	1.21 0.52 10	37.3 5.6 10	64.7 2.1 10	11.7 0.8 10	0.221 0.014 10	6.515 1.265 10	1.701 0.275 10	1.030 0.506 10	5.10 0.63 10	2.055 0.351 10	2.718 0.061 10	105.3 1.2 10	5.39 * 0.26 10	148.7 0.9 10	470.93 88.37 10
200 mg/kg	Mean SD N	73.5 ** 16.6 10	57.0 14.4 10	50.2 11.6 10	7.30 1.25 10	1.23 0.38 10	35.6 5.5 10	65.6 2.8 10	11.4 * 0.7 10	0.210 ** 0.009 10	7.262 0.885 10	1.730 0.290 10	0.854 0.271 10	4.94 0.60 10	2.082 0.318 10	2.732 0.054 10	105.8 0.8 10	5.39 * 0.37 10	148.9 0.6 10	471.04 62.42 10
1000 mg/kg	Mean SD N	86.4 17.8 10	61.0 10.0 10	40.3 6.4 10	6.70 1.16 10	0.98 0.25 10	38.3 4.4 10	65.3 3.7 10	11.4 * 0.8 10	0.212 ** 0.013 10	7.306 1.136 10	1.893 0.182 10	0.978 0.464 10	5.12 0.52 10	2.208 0.393 10	2.782 0.082 10	105.7 0.9 10	5.56 0.33 10	149.2 0.9 10	570.67 99.57 10

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

[G1] - Ancova/Anova & Dunnett(Log): * = p < 0.05

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

 

Table 10. Clinical chemistry females – day 92 relative to start date.

Sex: Female		ALP (U/L) [G]	ASAT (U/L) [G1]	ALAT (U/L) [G]	GGT (U/L) [G1]	Bilirub Total (umol/L) [G2]	Creatin ine (umol/L) [G1]	Total Protein (g/L) [G1]	Albumin (g/L) [G1]	Albumin/ Globulin   [G1]	Glucose Plasma (mmol/L) [G1]	Cholest erol (mmol/L) [G2]	Triglyc erides (mmol/L) [G1]	Urea (mmol/L) [G2]	PO4 (mmol/L) [G3]	Ca (mmol/L) [G2]	Cl  (mmol/L) [G2]	K (mmol/L) [G1]	Na (mmol/L) [G3]	T4 (ng/ml)
0 mg/kg	Mean SD N	49.4 15.0 10	79.7 10.8 10	57.2 21.6 10	7.70 0.82 10	0.80 0.25 10	40.4 4.1 10	69.1 2.8 10	13.7 1.6 10	0.248 0.031 10	5.450 0.804 10	1.414 0.315 10	0.771 0.231 10	6.20 0.66 10	2.209 0.260 10	2.783 0.050 10	105.9 0.7 10	5.62 0.46 10	146.6 1.3 10	468.30 93.41 10
40 mg/kg	Mean SD N	47.3 15.6 10	74.2 7.1 10	42.9 8.4 10	7.10 1.37 10	1.00 0.30 10	37.4 7.2 10	65.5 * 2.0 10	13.7 0.9 10	0.265 0.020 10	5.512 0.767 10	1.446 0.208 10	0.778 0.376 10	6.38 1.16 10	2.268 0.426 10	2.745 0.066 10	105.9 1.7 10	5.72 0.30 10	147.5 1.5 10	435.96 139.27 10
200 mg/kg	Mean SD N	41.1 8.7 10	71.1 5.7 10	44.7 14.1 10	6.60 0.84 10	1.18 0.81 10	43.0 5.9 10	69.5 3.2 10	13.9 1.9 10	0.250 0.034 10	5.568 0.738 10	1.456 0.461 10	0.813 0.341 10	6.94 1.57 10	2.094 0.271 10	2.777 0.045 10	105.4 1.2 10	5.74 0.33 10	146.6 1.5 10	474.06 82.02 10
1000 mg/kg	Mean SD N	38.0 5.3 10	77.9 7.7 10	41.7 10.7 10	7.00 1.25 10	1.07 0.59 10	42.8 6.0 10	69.7 3.5 10	14.5 1.7 10	0.262 0.025 10	5.110 0.562 10	1.496 0.290 10	0.869 0.283 10	7.28 0.67 10	2.141 0.315 10	2.793 0.087 10	104.4 * 1.1 10	5.75 0.28 10	145.4 0.7 10	388.54 110.98 10

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

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

[G2] - Kruskal-Wallis & Dunnett on Ranks

[G3] - Ancova/Anova & Dunnett

 

Table 11. Absolute organ weight males – day 92 relative to start date.

Sex: Male		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)   [G1]	Testes (g)   [G]	Epididymides (g) [G2]	Prostate (g)   [G]	Seminal vesicles (g)   [G]
0 mg/kg	Mean SD N	337.41 21.86 10	2.053 0.081 10	0.911 0.078 10	0.0441 0.0069 10	1.891 0.149 10	7.473 0.633 10	0.5049 0.0710 10	0.3266 0.0421 10	0.0099 0.0026 10	3.442 0.294 10	1.118 0.072 10	0.843 0.188 10	1.019 0.155 10
40 mg/kg	Mean SD N	339.70 32.37 10	1.991 0.044 10	0.936 0.076 10	0.0463 0.0073 10	1.832 0.194 10	7.792 0.731 10	0.4905 0.0585 10	0.2989 0.0611 10	0.0108 0.0020 10	3.278 0.261 10	1.103 0.172 10	0.883 0.118 10	1.185 0.163 10
200 mg/kg	Mean SD N	362.47 31.69 10	2.043 0.095 10	0.994 0.084 10	0.0487 0.0064 10	1.986 0.168 10	8.565 ** 0.948 10	0.5348 0.0539 10	0.3595 0.0886 10	0.0122 0.0031 10	3.520 0.370 10	1.189 0.141 10	0.860 0.108 10	1.038 0.203 10
1000 mg/kg	Mean SD N	365.57 22.67 10	2.045 0.073 10	0.953 0.080 10	0.0508 0.0068 10	1.904 0.146 10	8.335 * 0.754 10	0.5232 0.0633 10	0.3489 0.0639 10	0.0130 0.0050 10	3.389 0.348 10	1.215 0.091 10	0.929 0.122 10	1.100 0.162 10

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

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

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 12. Absolute organ weight females – 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) [G1]	Spleen (g) [G]	Thymus (g) [G]	Thyroid (g) [G]	Ovaries (g) [G2]	Uterus (g) [G1]
0 mg/kg	Mean SD N	216.93 12.73 10	1.920 0.071 10	0.739 0.077 10	0.0588 0.0107 10	1.373 0.092 10	5.429 0.514 10	0.4268 0.0470 10	0.2654 0.0496 10	0.0098 0.0021 10	0.0889 0.0136 10	0.9341 0.5332 10
40 mg/kg	Mean SD N	213.64 15.96 10	1.908 0.067 10	0.706 0.068 10	0.0642 0.0113 10	1.399 0.080 10	5.575 0.448 10	0.4479 0.0507 10	0.3210 0.0489 10	0.0115 0.0023 10	0.0931 0.0158 10	0.8480 0.2649 10
200 mg/kg	Mean SD N	222.49 15.51 10	1.904 0.081 10	0.693 0.062 10	0.0594 0.0076 10	1.428 0.135 10	5.713 0.658 10	0.4399 0.0575 10	0.2973 0.0623 10	0.0105 0.0022 10	0.0915 0.0163 10	0.8288 0.2644 10
1000 mg/kg	Mean SD N	214.08 10.86 10	1.874 0.037 10	0.673 0.065 10	0.0591 0.0073 10	1.316 0.099 10	5.464 0.279 10	0.4530 0.0568 10	0.3135 0.0556 10	0.0093 0.0018 10	0.0961 0.0262 10	0.6687 0.2828 10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

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

 

Table 13. Relative organ weight males – day 92 relative to start date.

		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) [G1]	Thymus rel.wgt (g/kg body wgt) [G]	Thyroid rel.wgt (g/kg body wgt) [G1]	Testes rel.wgt (g/kg body wgt) [G1]	Epididy rel.wgt (g/kg body wgt) [G1]	Prostate rel.wgt (g/kg body wgt) [G2]	Sem ves rel.wgt (g/kg body wgt) [G]
0 mg/kg	Mean SD N	337.41 21.86 10	6.108 0.476 10	2.700 0.155 10	0.1314 0.0235 10	5.619 0.504 10	22.15 1.16 10	1.502 0.228 10	0.969 0.119 10	0.0297 0.0092 10	10.197 0.457 10	3.321 0.238 10	2.503 0.554 10	3.027 0.481 10
40 mg/kg	Mean SD N	339.70 32.37 10	5.908 0.564 10	2.772 0.279 10	0.1363 0.0163 10	5.411 0.504 10	22.97 1.09 10	1.445 0.110 10	0.878 0.148 10	0.0322 0.0077 10	9.745 1.325 10	3.290 0.680 10	2.637 0.569 10	3.528 0.646 10
200 mg/kg	Mean SD  N	362.47 31.69 10	5.670 0.505 10	2.749 0.189 10	0.1347 0.0157 10	5.485 0.227 10	23.67 2.10 10	1.478 0.112 10	0.984 0.181 10	0.0336 0.0079 10	9.749 1.064 10	3.291 0.387 10	2.372 0.204 10	2.875 0.591 10
1000 mg/kg	Mean SD N	365.57 22.67 10	5.606 0.274 10	2.613 0.238 10	0.1399 0.0232 10	5.224 0.484 10	22.78 1.26 10	1.435 0.184 10	0.957 0.187 10	0.0360 0.0150 10	9.262 0.639 10	3.327 0.212 10	2.549 0.364 10	3.020 0.497 10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

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

 

Table 14. Relative organ weight females – 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) [G1]	Uterus rel.wgt (g/kg body wgt) [G2]
0 mg/kg	Mean SD N	216.93 12.73 10	8.865 0.357 10	3.402 0.230 10	0.2710 0.0463 10	6.330 0.237 10	25.05 2.15 10	1.967 0.192 10	1.233 0.268 10	0.0451 0.0090 10	0.4098 0.0582 10	4.267 2.413 10
40 mg/kg	Mean SD N	213.64 15.96 10	8.970 0.659 10	3.315 0.349 10	0.3017 0.0565 10	6.571 0.480 10	26.15 1.92 10	2.101 0.225 10	1.515 0.290 10	0.0539 0.0104 10	0.4367 0.0725 10	3.976 1.241 10
200 mg/kg	Mean SD N	222.49 15.51 10	8.581 0.452 10	3.116 * 0.188 10	0.2675 0.0325 10	6.415 0.373 10	25.76 3.06 10	1.980 0.241 10	1.344 0.304 10	0.0470 0.0088 10	0.4108 0.0679 10	3.775 1.379 10
1000 mg/kg	Mean SD N	214.08 10.86 10	8.770 0.384 10	3.140 0.210 10	0.2758 0.0283 10	6.150 0.390 10	25.57 1.68 10	2.114 0.207 10	1.469 0.276 10	0.0436 0.0090 10	0.4461 0.1018 10	3.137 1.366 10

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

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

[G2] - Kruskal-Wallis & Dunnett on Ranks

Table 15. Thyroid hormone levels in males and females – Day 91 and 92 relative to start date, respectively.

		Male	Male	Female	Female
		TSH (pg/ml) [G]	T4 (ng/mL)	TSH (pg/ml) [G1]	T4 (ng/mL)
0 mg/kg	Mean SD N	2419.25 728.82 10	532.37 103.37 10	2977.77 551.55 10	468.30 93.41 10
40 mg/kg	Mean	2406.56	470.93	3604.39	435.96
	SD	736.17	88.37	1405.95	139.27
	N	10	10	10	10
200 mg/kg	Mean	1947.21	471.04	2716.86	474.06
	SD	505.32	62.42	709.22	82.02
	N	10	10	10	10
1000 mg/kg	Mean	2498.93	570.67	2216.54 *	388.54
	SD	994.38	99.57	296.92	110.98
	N	10	10	10	10

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

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

Table 16. Historical control data for TSH in serum samples.

	Males			Females
Study type	OECD 443	OECD 443	OECD 408	OECD 443	OECD 443	OECD 408
Approx. age of rats	22 weeks	13 weeks	19 weeks	19 weeks	13 weeks	19 weeks
Mean	2661	3872	2969	1923	983	3683
SD	1174.6	1047.5	798.6	692.0	567.6	1134.2
CV	44.1%	27.1%	26.9 %	36.0%	57.7%	30.8 %
Count	10	8	10	10	9	10

Conclusions:

In this GLP complaint oral repeated dose toxicity study, performed according to OECD 408, the no-observed-adverse effect level (NOAEL) was placed at the highest level tested, namely 1000 mg/kg bw/day because the test substance did not induce any toxicologically relevant changes in any test group.
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 this GLP compliant 90 day gavage study performed according to OECD 408, the safety of ZBEC was examined in Wistar rats of both sexes. ZBEC was administered to the rats by daily oral gavage as a suspension in corn oil during 13 weeks at levels of 0, 40, 200 and 1000 mg/kg body weight/day. 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. Haematology, clinical chemistry (including thyroid hormones TSH and T4) 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.

The administration of ZBEC was well tolerated at all dose levels, and did not induce any relevant changes in general condition, growth, feed or water intake, neurobehavioral observations, ophthalmoscopy, urinalysis, organ weights or in macroscopy and microscopy of organs and tissues. A few differences with the control reached statistical significance in the high-dose group, but were not considered to be adverse as discussed below: In females of the high dose group, red blood cell count, haemoglobin concentration and packed cell volume were somewhat lower than in controls. Because these changes were only slight (about -6%) they were not considered to be adverse. Furthermore, the percentage of lymphocytes was increased and the percentage of neutrophils was decreased in males of the high-dose group. Because these findings were within the range of historical control data and not reflected by significant changes in the absolute lymphocyte and neutrophil counts, they were not considered to be adverse. Albumin and the albumin/globulin ratio were decreased in males of the mid- and high-dose groups and chloride concentration was decreased in high-dose females. Because these changes were within the range of historical control data and not corroborated by any effects on relative organ weight or pathology, they were not considered to be adverse. TSH levels were slightly decreased in females of the high-dose group. This slight decrease in TSH levels was not corroborated by noticeable changes in T4 levels, growth, thyroid weight or pathology. Moreover the values were well within the range of historical control data and this variable is known to show considerable variation. Therefore the decrease in TSH levels in high-dose females was considered to be a chance finding.

Because ZBEC did not induce any relevant changes in any test group, the no-observed-adverse effect level (NOAEL) was placed at the highest level tested, namely 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant, OECD 408 study

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 - 90 day in rat

In a GLP compliant 90 day gavage study performed according to OECD 408, the safety of ZBEC was examined in Wistar rats of both sexes (Triskelion, 2018). ZBEC was administered to the rats by daily oral gavage as a suspension in corn oil during 13 weeks at levels of 0, 40, 200 and 1000 mg/kg body weight/day. 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. Haematology, clinical chemistry (including thyroid hormones TSH and T4) 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.

The administration of ZBEC was well tolerated at all dose levels, and did not induce any relevant changes in general condition, growth, feed or water intake, neurobehavioral observations, ophthalmoscopy, urinalysis, organ weights or in macroscopy and microscopy of organs and tissues. A few differences with the control reached statistical significance in the high-dose group, but were not considered to be adverse as discussed below: In females of the high dose group, red blood cell count, haemoglobin concentration and packed cell volume were somewhat lower than in controls. Because these changes were only slight (about -6%) they were not considered to be adverse. Furthermore, the percentage of lymphocytes was increased and the percentage of neutrophils was decreased in males of the high-dose group. Because these findings were within the range of historical control data and not reflected by significant changes in the absolute lymphocyte and neutrophil counts, they were not considered to be adverse. Albumin and the albumin/globulin ratio were decreased in males of the mid- and high-dose groups and chloride concentration was decreased in high-dose females. Because these changes were within the range of historical control data and not corroborated by any effects on relative organ weight or pathology, they were not considered to be adverse. TSH levels were slightly decreased in females of the high-dose group. This slight decrease in TSH levels was not corroborated by noticeable changes in T4 levels, growth, thyroid weight or pathology. Moreover the values were well within the range of historical control data and this variable is known to show considerable variation. Therefore the decrease in TSH levels in high-dose females was considered to be a chance finding.

Because ZBEC did not induce any relevant changes in any test group, the no-observed-adverse effect level (NOAEL) was placed at the highest level tested, namely 1000 mg/kg bw/day.

Justification for classification or non-classification

Classification of ZBEC for toxic effects upon repeated exposure is not warranted in accordance with EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.