Bis(dibutyldithiocarbamato-S,S')copper

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Aug 2021 - 15 Dec 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dibutyldithiocarbamato-S,S')copper

EC Number:

237-695-7

EC Name:

Bis(dibutyldithiocarbamato-S,S')copper

Cas Number:

13927-71-4

Molecular formula:

C18H36CuN2S4

IUPAC Name:

bis(dibutyldithiocarbamato-S,S')copper

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) Sprague-Dawley rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks
- Weight at at initiation of dosing: between 261.3 and 372.1 g (males) and between 192.0 and 266.2 g (females)
- Fasting period before study: not reported
- Housing: The animals were group housed, two or three animals of the same sex and same dosing group together, in polycarbonate cages with stainless steel lids containing autoclaved sawdust. Animals were separated during designated procedures/activities, as deemed appropriate by the study director and/or clinical veterinarian. Cages were arranged on the racks in group order. For psychological/environmental enrichment, animals were provided with rat hut and nylabone.
- Diet: ad libitum; SSNIFF rat/mouse pelleted maintenance diet, sterilized by irradiation
- Water: ad libitum; Municipal tap water filtered with a 0.22 µm filter
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
- Contamination: It was considered that there were no known contaminants in the feed and water that interfered with the objectives of the study

ENVIRONMENTAL CONDITIONS (targeted conditions)
- Temperature: 20°C to 24°C
- Humidity: 30% to 70%
- Air changes: Approximately 8 to 15 filtered, non-recycled air changes per hour
- Photoperiod: 12 hours light and 12 hours dark

IN-LIFE DATES: From 12 Sep 2021 To 15 Dec 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

(0.5% (w/w) aqueous methylcellulose, 4000 cPs)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared daily, on the day of each administration:
Control Item: Solution; Frequency of preparartion: as stated in SOP; Delivery conditions: at room temperature
Test Item: Suspension; Frequency of preparartion: daily; Delivery conditions: at room temperature
The control and test item dose formulations were stirred continuously for at least 15 minutes before dosing and during dosing and were maintained under delivery conditions (at room temperature)

VEHICLE
- Concentration of test substance in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Remarks:

Dose formulation samples were collected for analysis of concentration and homogeneity on as day 1, week 4, 8 and 13. See for more details Table 1 under 'Any other information on materials and methods incl. tables'

Details on analytical verification of doses or concentrations:

Analyses were performed by UV-Vis spectrophotometry using a validated analytical procedure (Test Facility Study No. 44631 AHS/44630 VAA).

Acceptance Criteria:
For concentration: Individual sample concentration of ± 15%, mean sample concentration results within or equal to ± 15% of theoretical concentration
For homogeneity, Relative Standard Deviation (RSD) of concentrations = 10% for each Group.

Stability analyses performed previously in conjunction with study No. 44630 VAA demonstrated
that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 4

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 based on information provided by the Sponsor. Test Facility Study No. 44633 RSR demonstrated that the dose of 1000 mg/kg bw/day could be considered such as the NOAEL (No Observed Adverse Effect Level) in the case of rat species.
- Fasting period before blood sampling for clinical biochemistry: Yes, A minimum of 14 hours before sample collection for clinical chemistry and uranalysis.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality Check: At least twice daily (at the beginning and end of the working day) beginning upon arrival through termination (except on days of receipt and necropsy where frequency was at least once daily). Animals were observed within their cage unless necessary for identification or confirmation of possible findings.
Cage side Observations: At least once daily; from at least Week -2. Animals were observed within their cage unless necessary for identification or confirmation of possible findings.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Post-dose Observations: Daily, after completion of dosing of the last animal in the animal room on the days of dosing. Animals were observed within their cage unless necessary for identification or confirmation of possible findings. Particular attention was paid to signs such as piloerection.
Detailed Clinical Observations: Weekly; from at least Week -2 and throughout the study or before unscheduled euthanasia. Animals were removed from the cage.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Week -2 and throughout the study. Last body weight before terminal fasting or before unscheduled euthanasia.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly for each cage of animals; from Day 1 and throughout the study. Quantitatively measured. Calculated per animal and per day. When one of the animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of food consumption.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once pretest (including alternate animals). At the end of the dosing period for the control (group 1) and high-dose study (group 4) animals only.
The pupils of the animals were dilated with tropicamide (Mydriaticum). After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy

BLOOD SAMPLING
HAEMATOLOGY: Yes
Blood samples for heamatology, clinical chemistry, coagulation and thyroid hormone were collected and the end of the dosing period via abdominal aorta immediately prior to necropsy.
- Anaesthetic used for blood collection: Yes, i.p. injection of sodium pentobarbital
- Animals fasted: Yes, overnight (at least 14 hours)
- How many animals: All main study animals.
- See Table 2 for Heamatology parameters, Table 3 for Coagulation, Table 4 for Clinical Chemistry parameters under "Any other information on materials and methods incl. tables".

THYROID HORMONES:
- Time schedule: End of dosing period, blood was collected, at a comparable time in the first half of the morning (between 7.5 and 10 a.m.) via jugular vein
- Animals fasted: Yes, overnight (at least 14 hours)
- How many animals: All main study animals.
The levels of the thyroid hormones (T3 and T4) was determined by LC MS/MS method and those of Thyroid Stimulating Hormone (TSH) by Luminex MAP® technology.

URINANALYSIS: Yes
- Time schedule for collection of urine: At the end of the dosing period, overnight in individual metabolism cages, prior to euthanasia.
- Animals fasted: Yes, animals are fasted during the collection of urine.
- How many animals: All study animals
- Parameters in Table 5 "Any other information on materials and methods incl. tables" were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the treatment period.
- Dose groups that were examined: All study animals.
- Battery of functions tested:
• in the cage: touch escape,
• in the hand: fur appearance, salivation, lacrimation, piloerection, pupil size (presence of myosis or mydriasis), exophthalmia, reactivity to handling,
• in the standard arena (2-minute recording): grooming, palpebral closure, tremors, twitches, convulsions, arousal (hypo- and hyper-activity), ataxia, hypotonia, gait, posture, stereotypy, behavior, breathing, defecation and urination.
• reactivity to the following manipulation and stimulis was evaluated: touch response, visual stimulus, pupillary reflex, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, forelimb grip strength,
• at the end of observation: rectal temperature.

For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.


IMMUNOLOGY: No

ESTROUS STAGE DETERMINATION: Yes
- Time schedule: end of treatment on day of necropsy, a vaginal smear was taken to determine the stage of estrus (stained with methylene blue).
- How many animals: All females

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, on all animals
-Necropsy: All study animals (including a prematurely euthanized female from Group 4 (At 1000 mg/kg bw/day)) were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues
-Organ weights: The organs detailed in the Table 6 under "Any other information on materials and methods incl. tables" were weighed at necropsy. Paired organs were weighed together. For gross abnormalities, in addition to the combined weight, the weight of each organ of a pair was taken and entered as a tissue comment.
Organ weight as a percent of body weight (using the terminal body weight) and organ weight as a percent of brain weight were calculated.

HISTOPATHOLOGY: Yes
- Tissue Collection and Preservation: Representative samples of tissues identified in Table 7 under "Any other information on materials and methods incl. tables" were collected and preserved in 10% neutral buffered formalin, unless otherwise indicated.
Tissues identified in Table 7 (except optic nerves and eyes with Harderian glands since there were no changes observed during ophthalmological examinations) were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin
- Microscopic Evaluation
Tissues as detailed in Table 7 (except optic nerves) were histopathologically evaluated by a board-certified veterinary pathologist. Special stains could be used at the discretion of the pathologist to further characterize lesions and changes identified during routine evaluation of individual animals. Any special stains were documented in the individual animal data.

Statistics:

All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated under "Detailed Information on Statistical Analysis" under the section "Any other information on materials and methods incl. tables" according to sex and occasion.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs considered to be related to test item administration.
Clinical signs observed during the study included missing, short/broken teeth, scabs or wounds, tail bent, soiled animal, thin fur and/or alopecia. They were isolated and commonly observed in untreated Group-housed laboratory rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 1000 mg/kg bw/day, one female, was prematurely euthanized for humane reasons on Day 57. Following gavage difficulties on Day 55 where the animal struggled during gavage, treatment was stopped on Day 56 to allow recovery. This female showed decreased activity on Day 55 post-dosing then on Day 57, erected fur on Days 56 and 57, a hunched posture from Day 55 to Day 57, had abdominal breathing on Days 55 and 56, a thin appearance and was prostrate on Day 57. The animal lost 24.2 g between Days 50 and 57. Macroscopic observations at necropsy included thinned esophagus, enlarged pericardium as well as adhesions of pericardium to ribs, lungs, thymus and diaphragm and accentuated lobular pattern of the liver. Black thick deposits and translucent liquid were found in the thoracic cavity.
Microscopic findings included acute or mixed cell inflammation affecting the lung pleura, pericardium and the serosa of esophagus and aorta. The microscopic findings correlated with the enlarged pericardium and the adhesions to thoracic organs observed at necropsy.
The macroscopic and microscopic findings were consistent with a dose related injury which was considered the cause of death. Therefore this death was unrelated to the test item.
There were no other unscheduled deaths during the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 1000 mg/kg bw/day, mean body weights were slightly lower than controls in both male (down to -1.4% vs controls) and female (down to -7.8% vs controls) animals, at the end of the treatment period. Although a test item related effect was not excluded, all rats gained weight and this change, which had no effect on the overall animals’ condition, was not considered as adverse. See Table 8 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slightly reduced food consumption observed for females at 300 and 1000 mg/kg bw/day was consistent with body weight changes. See Table 9 under "Any other information on results incl. tables".

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmological abnormalities in any animal at 1000 mg/kg bw/day at the end of the treatment period, when compared to controls.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in hematology parameters attributed to test item.
The increase in neutrophil count observed in all treated males compared to controls was not dose-related and values within the normal background variations for this species. This change was therefore considered as incidental. See Table 10 under "Any other information on results incl. tables".
When compared to control mean values, there were no test item-related changes in coagulation parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with control values, there were decreases in triglyceride concentrations. As these changes lack dose relationship, had no microscopic correlates, they were not considered to be test item-related.
The differences between control and test item-related Groups, including those reaching statistical significance, were of minimal magnitude, seen with no dose-relationship, or in the range of historical control data, and were therefore not considered to be test item-related.
See Table 11 under "Any other information on results incl. tables".

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The differences between control and test item-related Groups, including those reaching statistical significance, were of minimal magnitude, seen with no dose-relationship, or in the range of historical control data, and were therefore not considered to be test item-related. See Table 12 under "Any other information on results incl. tables".

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared to controls, there was a decrease in mean pH values at all dose levels in males. It was more pronounced at 1000 mg/kg bw/day, reaching approximately -10% vs. controls. These changes were, therefore, not considered as adverse. There were no other changes attributed to test item in urinary parameters.
See Table 13 under "Any other information on results incl. tables".

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls, there were no changes in behavioral functional observation battery attributed to test item in any Group at the end of the treatment period. There were no obvious changes in body temperature or motor activity in any Group.
The few differences, even those statistically significants observed were either isolated, of low magnitude or not dose-related and therefore considered as incidental.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related organ weight changes were noted. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed, including those statistically significant, were considered incidental and unrelated to administration of test item.
See Table 14 under "Any other information on results incl. tables".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of test item.
See Table 15 under "Any other information on results incl. tables".

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals or with isolated occurrence in the treated animals. Therefore, they were considered unrelated to administration of test item.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Dose formulation analysis

The dose formulations prepared on Day 1, in Weeks 4, 8 and 13, were all found within the acceptance criteria (measured concentrations at ± 15% of the theoretical concentrations).

No test item was found in the control dose formulation.

Dose formulation homogeneity was also demonstrated as the Relative Standard Deviation (RSD) was found to be ≤ 10% for each tested Group on Day 1.

 

Table 9: Summary of Mean Body Weight and Mean Body Weight Change

Dose level (mg/kg bw/day)	Males				Females
	0	100	300	1000	0	100	300	1000
Body weight (g)
Day 1	322.1	331.9	325.5	329.8	226.9	224.4	221.2	216.9
vs. controls (%)	-	+3.0	+1.1	+2.4	-	-1.1	-2.5	-4.4
Day 29	474.8	479.7	473.6	475.0	281.0	277.3	261.7	259.0
vs. controls (%)	-	+1.0	-0.2	+0.1	-	-1.3	-6.9	-7.8
Day 57	562.2	569.4	552.9	549.6	311.9	302.9	284.0	282.6
vs. controls (%)	-	+1.3	-1.7	-2.2	-	-2.9	-8.9	-9.4
Day 91	606.2	618.8	599.8	597.7	322.8	316.3	299.0	297.6
vs. controls (%)	-	+2.1	-1.1	-1.4	-	-2.0	-7.4	-7.8
Body weight Change (g)
Day 1 to Day 91	284.1	286.9	274.2	267.9	95.8	92.0	77.8	80.2
vs. controls (%)	-	+1.0	-3.5	-5.7	-	-4.0	-18.8	-16.3
- : not applicable

 

Table 9: Summary of Mean Food Consumption Change

Dose level (mg/kg bw/day)	Males				Females
	0	100	300	1000	0	100	300	1000
Food Consumption (g/day)
Day 22 to Day 29	-	-	-	-	23.8	20.3*	19.5***	23.0
vs. controls (%)	-	-	-	-	-	-14.6	-18.0	-3.4
Day 29 to Day 36	-	-	-	-	21.7	22.0	19.3*	19.0*
vs. controls (%)	-	-	-	-	-	+1.2	-11.3	-12.7
Day 57 to Day 64	-	-	-	-	21.0	21.6	17.4***	19.4
vs. controls (%)	-	-	-	-	-	+3.0	-17.4	-7.8
Day 1 to Day 91	31.6	32.8	31.9	32.5	21.3	21.8	19.5	19.7
vs. controls (%)	-	+3.7	+0.9	+2.9	-	+2.2	-8.5	-7.7
Statistically significant from controls: *: p < 0.05; ***: p < 0.001; -: no changes

 

Table 10: Summary of Hematology Changes on Day 92

Dose level (mg/kg bw/day)	Males					Females
	0	100	300	1000	HCD	0	100	300	1000	HCD
Parameter
NEUT	0.82	1.34	1.46*	1.18	[0.53-3.87]	-	-	-	-	-
vs. controls (%)	-	+64.6	+79.0	+45.0	-	-	-	-	-	-
Statistically significant from controls: *: p < 0.05; -: no changes; NEUT: neutrophils ; HCD: Historical Control Data, [min.; max.].

 

Table 11: Summary of Clinical Chemistry Changes on Day 92 (males) or 93 (females)

Dose level (mg/kg bw/day)	Males					Females
	0	100	300	1000	HCD	0	100	300	1000	HCD
Parameter
TRIG	0.43	0.33	0.23*	0.24*	[0.22-1.33]	0.37	0.24	0.17***	0.19**	[0.22-0.67]
vs. controls (%)	-	-24.7	-46.4	-45.5	-	-	-36.8	-53.5	-48.9	-
K	3.65	3.76	3.88	3.97	[3.30-7.29]	-	-	-	-	-
vs. controls (%)	-	+3.1	+6.3	+8.9	-	-	-	-	-	-
CA	2.54	2.52	2.48*	2.45**	[2.43-2.93]	-	-	-	-	-
vs. controls (%)	-	-1.0	-2.6	-3.4	-	-	-	-	-	-
PHOS	-	-	-	-	-	1.50	1.91**	1.73	1.72	[1.32-4.25]
vs. controls (%)	-	-	-	-	-	-	+27.7	+15.5	+15.0	-
CREA	-	-	-	-	-	37.94	39.42	38.79	35.65	[27.20-54.98]
vs. controls (%)	-	-	-	-	-	-	+3.9	+2.2	-6.0	-
CHOL	-	-	-	-	-	2.13	1.83	1.77	1.66	[0.77-2.32]
vs. controls (%)	-	-	-	-	-	-	-14.1	-16.9	-22.0	-
ALP	-	-	-	-	-	27	37**	33	34	[50-200]
vs. controls (%)	-	-	-	-	-	-	+35.8	+19.0	+22.5	-
ASAT	-	-	-	-	-	76	94	94	106	[52-147]
vs. controls (%)	-	-	-	-	-	-	+24.7	+23.7	+39.9	-
ALAT	-	-	-	-	-	33	39	37	47	[15-72]
vs. controls (%)	-	-	-	-	-	-	+18.3	+11.4	+41.1	-
Statistically significant from controls: *: p < 0.05; **: p < 0.01; ***: p < 0.001; -: no changes; TRIG: Triglyceride concentration; K: Potassium; CA: Calcium; PHOS: Phosphorus; CREA: Creatinine; CHOL: Cholesterol; ALP: Alkanine Phosphatase; ASAT: Aspartate Aminotransferase; ALAT: Alanine Aminotransferase; HCD: Historical Control Data, [min.; max.].

 

Table 12: Summary of Thyroid hormones on Day 87/88

Dose level (mg/kg bw/day)	Males					Females
	0	100	300	1000	HCD	0	100	300	1000	HCD
T3 (ng/mL)	0.3	0.45*	0.38	0.45**	[0.15-0.53]	0.48	0.46	0.45	0.45	[0.27-0.70]
vs. controls (%)	-	51.2	28.1	52.9	-	-	-4.2	-6.9	-6.3	-
T4 (ng/mL)	46.88	50.99	46.55	44.16	[30.19-81.21]	25.15	28.66	28.26	28.3	[13.81-41.48]
vs. controls (%)	-	8.8	-0.7	-5.8	-	-	14	12.4	12.5	-
TSH (pg/mL)	902	1121	1506	922	[489-15000]	444	552	495	448	[196-2030]
vs. controls (%)	-	24.3	67.1	2.3	-	-	24.4	11.6	1.1	-
Statistically significant from controls: *: p < 0.05; **:p < 0.01; -: not applicable.

 

Table 13: Summary of Urinary pH Changes on Day 92

Dose level (mg/kg bw/day)	Males				Females
	0	100	300	1000	0	100	300	1000
Parameter
pH	7.6	7.2*	7.2	6.8***	-	-	-	-
vs. controls (%)	-	-5.3	-4.6	-9.9	-	-	-	-
Statistically significant from controls: *: p < 0.05; ***:p < 0.001; - : no changes.

 

Table 14: Summary of statistically changes in Organ Weight (g)

Dose level (mg/kg bw/day)	Males				Females
	0	100	300	1000	0	100	300	1000
Brain					1.98	2.02	2.03	2.01
Mean % of bodyweight	-	-	-	-	0.64483	0.068827	0.74006**	0.72423*
Heart	-	-	-	-	1.09	1.06	0.99	0.99
Mean % of brain weight	-	-	-	-	55.5	52.6	48.95*	49.70*
Liver	-	-	-	-	8.22	7.57	7.23	7.22
Mean % of brain weight	-	-	-	-	417	376	356*	360*
Statistically significant from controls: *: p < 0.05; **:p < 0.01; -: not applicable.

 

Table 15: Summary of Macroscopic findings (not test item-related)

 

Dose level (mg/kg bw/day)	Males				Females
	0	100	300	1000	0	100	300	1000
Esophagus - thinned								1/10^a
Harderian glands - black discoloration						1/10		1/10^a
Heart - adhesion(s) to ribs and enlarged								1/10^a
Kidney - dilated pelvis	1/10			1/10
Kidney - irregular color		1/10
Liver - accentuated lobular pattern	2/10	2/10	4/10	2/10		1/10		3/10
Spleen - deformation		1/10
Skin - scabs							1/10
Stomach - white discoloration	1/10				2/10		1/10
Stomach - black discoloration						1/10		1/10
Stomach - red discoloration							1/10
Thoracic cavity - black deposit								1/10
Thoracic cavity - translucent content								1/10
Thyroid glands - reduced in size						1/10
Thymus - red discoloration	2/10
Uterus – Dilatation, translucent content					1/10	4/10	4/10	3/10
Urinary bladder - dilatation				1/10

 

Applicant's summary and conclusion

Conclusions:

Daily oral administration of Copper dibutyldithiocarbamate to Sprague-Dawley rats for 13 weeks at levels of 100, 300 and 1000 mg/kg bw/day was well tolerated.
Minimal changes consisting of decreased triglycerides (both genders) at all dose levels, decreased pH (males) at all dose levels, were no considered as adverse.
In the absence of adverse findings and test item-related observations in microscopic evaluation, the No Observed Adverse Effect Level (NOAEL) was set at 1000 mg/kg bw/day.

Executive summary:

A repeated dose toxicity study was performed in rats according to OECD TG 408 and in accordance with GLP principles. The objective of this study was to determine the potential toxicity of Copper Dibutyldithiocarbamate (CDBC), when given by oral (gavage) route for 13 weeks to male and female rats. 

Methods

The study design was as follows:

Experimental Design

Group Number	Test Material	Dose Level (mg/kg bw/day)	Dose Volume (mL/kg bw)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	Vehicle	0	10	0	10	10
2	CDBC	100	10	10	10	10
3	CDBC	300	10	30	10	10
4	CDBC	1000	10	100	10	10

Vehicle: 0.5% (w/w) aqueous methylcellulose (4000 cPs).

Animals were treated daily during 91 days (males) and 92 days (females).

Four Groups of Sprague Dawley rats, each including ten animals/sex were treated by the oral (gavage) route with the vehicle (0.5% (w/w) aqueous methylcellulose, Group 1) or the test item (Copper dibutyldithiocarbamate) as a suspension in the vehicle at 100, 300 or 1000 mg/kg bw/day (Groups 2, 3 and 4, respectively) for 91 (males) or 92 (females) days. A constant dosage volume of 10 mL/kg/day was used.

The assessment of toxicity was based on the following parameters and end points : mortality, clinical observations, body weights, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry and urinalysis), functional observation battery and thyroid hormones analysis. On completion of the treatment period, animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed. Microscopic examination on a representative selection of tissues was performed.

 

Results

The dose formulations prepared on Day 1, in Weeks 4, 8 and 13, were all found within the acceptance criteria (measured concentrations at ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.

There were no test item-related deaths, clinical signs, functional observation battery parameters (including body temperature) changes, ophthalmologic findings, hematological and coagulation parameters changes in any Group during the study.

At 300 and 1000 mg/kg bw/day, mean body weights (down to -7.8% in females at 1000 mg/kg bw/day) were slightly lower than controls, at the end of the treatment period. Although a test item related effect was not excluded, all rats gained weight and this change, which had no effect on the overall animals’ condition, was not considered as adverse. A slightly reduced food consumption observed for females at 300 and 1000 mg/kg bw/day was consistent with body weight changes. 

At 300 and 1000 mg/kg/day, there was a decrease in triglyceride levels in males [-46.4% and -45.5% (p < 0.05) vs. controls at 300 and 1000 mg/kg bw/day, respectively] and in females [-53.5% (p < 0.001) and -48.9% (p < 0.01) vs. controls at 300 and 1000 mg/kg/day, respectively]. All values remained, however, within the normal background range for this species and these changes were not considered as adverseWhen compared with controls, there was a decrease in mean pH values at all dose levels in males, more pronounced at 1000 mg/kg bw/day [approximately -10% vs. controls (p < 0.001)]. These results were not considered as adverse.

Thyroid hormones changes were of minimal magnitude and seen with no dose-relationship, and were therefore not considered to be test item-r elated.

No test item-related changes in organ weight, macroscopic and microscopic findings were noted in the study.

 

Conclusion

Daily oral administration of Copper dibutyldithiocarbamate to Sprague-Dawley rats for 13 weeks at levels of 100, 300 and 1000 mg/kg bw/day was well tolerated.

Minimal changes consisting of slightly decreased body weight gains and/or decreased food consumption at 300 and 1000 mg/kg bw/day, decreased triglycerides (both genders) at all dose levels, decreased pH (males) at all dose levels, were no considered as adverse.

In the absence of adverse findings and test item-related observations in microscopic evaluation, the No Observed Adverse Effect Level (NOAEL) was set at 1000 mg/kg bw/day.