Registration Dossier

Administrative data

Description of key information

A 28 -day repeated toxicity study in available on Copper dibutyldithiocarbamate. Based on this study, the no-observed adverse effect level (NOAEL) is of 1000 mg/kg/day in both males and females because no relevant adverse systemic effects were observed at the highest dose of 1000 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan Charles River Laboratories International, Inc. (Atsugi Production Facility)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: Males: 319~368g, Females: 212~249 g

- Housing: Male and female, 1 each, were housed per cage in the mating period. 1 litter was housed during the lactation period, and 1 animal was housed in the other periods, including the quarantine period.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum ; Polycarbonate water bottles (200 and 700 mL) were used, and replaced on the date of grouping and subsequently at a frequency of 1 time within 7 days from the administration start date.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0~25.0°C
- Humidity (%): 35.0~75.0
- Air changes (per hr): 6~20 times/hour, all-fresh air supply
- Photoperiod (hrs dark / hrs light): 12 hours/day (7:00~19:00)
Route of administration:
oral: gavage
Details on route of administration:
Administration was made using a disposable syringe with a feeding tube attached. The administration solution was used while thoroughly agitating with a stirrer.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.1% w/v Tween 80-spiked 0.5% w/v CMC-Na aqueous solution (Tween 80: Kanto Kagaku K.K., Lot No.: 807X1315, CMC-Na: Wako Pure Chemical Industries, Ltd., Lot No.: SDL2940)
Details on oral exposure:
This was 10 mL/kg, and the fluid volume was calculated for each individual based on the weight measured on the most recent day.

Preparation was performed in the preparation room under illumination with UV rays cut out.
(100 mg/mL preparation fluid)
The tested substance was weighed out to the stipulated weight, and pulverized in a mortar and pestle. The medium was then gradually added, and then was collected and suspended by ultrasonic processing (approx. 30 minutes, ultrasonic cleaner, W-115, Honda Electronics Co., Ltd.). It was then graded up to a concentration of 100 mg/mL using a graduated cylinder.
(4 and 200 mg/mL preparation fluid)
The 4 and 20 mg/mL fluid were prepared by diluting the 100 mg/mL fluid with the medium.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (n=3) taken from three points in the Administration Solution at initial preparation (top layer, middle layer, and bottom layer) were analysed according to the methods of Item 7.7. As a result, the average concentration (actual measurement: 91.0~93.8%, within ±10% of set concentration) and uniformity (actual measurements: 0.2%~0.9%, within 10% C.V. of the analysis values of the top layer, middle layer, and bottom layer), were confirmed as being within the allowable range (Item 12.4).
Duration of treatment / exposure:
Males: A total of 42 days from 14 days before mating, through the mating period, and up to the day before autopsy.
Females : This was the 14 days before mating, through the mating period, Gestation period, and parturitions, up to the 4th day of lactation (treating the date of parturition as lactation day 0). However, this was up to 25 days after confirmation of mating for non-pregnant animals.
Satellite animals (females): Up to the 42nd day without mating.
Frequency of treatment:
daily
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
females : 12 / dose
males : 7/ dose (control and high dose groups), 12/dose (low and medium dose)
satelite groups (control and high dose groups): 5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
A 14-day recovery period after the end of the Dosing Period was set for 5 males and females, respectively, of the control group and 1000 mg/kg group. However, the females were set as a satellite where mating was not performed.
The following items were examined. The recording of the dates for the males and female satellite animals treated the administration start date as Day 1, days 1~7 as Week 1, and from day 43 onward as the recovery period. In addition, for the female test animals, the date of administration start was treated as day 0 before mating, the date of mating establishment as day 0 of gestation, and the day of gestation completion as day 0 of lactation.
Positive control:
no
Observations and examinations performed and frequency:
Detailed Symptom Observation
(1) Observation in Home Cage
The animals were observed in the home cage for 1 minute in a resting state.
Examination Items: Tremors, clonic seizures, Tonic convulsions, respiration

(2) Observation during Handling
The animal torsos were gently grasped from the back, and removed from the cage for observation.
Examination Items: Ease of removal from cage, response to handling, aggressiveness, skin (injuries, skin colour), fur (soiling of fur), eyes (eyeball protrusion, closed eyelid state), mucosa (colour of conjunctiva), excreta, lacrimation, ptyalism, piloerection, pupil diameter

(3) Observation in Open Field
Observation was performed for 2 minutes with the animals in a rested state in the centre of an open field (Before placing the animals in the open field, wipe the floor with strongly-wrung wet cloth).
Examination Items: Standing, awareness, urination, defecation, posture/stance, respiration, motor coordination, gait abnormalities, tremors, clonic seizures, Tonic convulsions, normal behaviour, abnormal behaviour

Functional Examination
(1) Responsiveness to Stimuli
This was examined in an open field.
Examination Items: Response to approach, response to contact, auditory response, tail-pinch response, airborne righting reflex.

(2) Grip Strength Measurement
This was measured with a digital force gauge (Type: DPS-5).
Examination Items: Foreleg grip strength, hind leg grip strength

Measurement of Spontaneous Movement
A spontaneous movement measurement device (SUPERMEX, Muromachi Kikai Co., Ltd.) was used. Both male and female animals were moved to a polycarbonate cage (265W×426D×200H mm, Tokiwa Kagaku, K.K.) after the completion of the post-administration observation of week 6 (day 41), and cage domestication was performed (individual care). This was replaced with a new polycarbonate cage prior to measurement, and measurement performed for 1 hour. Furthermore, the measurements were totalled every 10 minutes from the start of measurement.

Body Weight
The male testing animals and male recovery animals were measured on days 1, 8, 15, 22, 29, 36, and 43, and the recovery animals further measured on days 50 and 56. The female satellite animals were measured at the same frequency as the male recovery animals. The female test animals were measured on days 0, 7, and 14 before mating, the mated females on days 0, 7, 14, and 20 of gestation, and the pregnant females on days 0 and 4 of lactation. The measurement used an electronic platform scale (EB-3200S: Shimadzu Corporation, PB3002-S: Mettler-Toledo International, Inc.).

Feed Intake
The male test animals and male recovery animals were measured on days 1~8, 8~15, 22~29, 29~36, 36~40, and 43~50, and the female satellite animals were measured on days 1~8, 8~15, 15~22, 22~29, 29~36, 36~42, 43~50, and 50~56. The female test animals were measured at the same frequency as the body weight measurement. However, measurement was not performed while cohabiting in the mating period (neither males nor females were measured as all cases were mating on days 15~22). The measurement of males after mating with a female had been confirmed was started from the nearest measurement date. The measurement used an electronic platform scale (EB-3200S: Shimadzu Corporation, PB3002-S: Mettler-Toledo International, Inc.). All measured the tared weight on a per-cage basis, and the average daily food intake was calculated per-animal for each measurement period.

Haematological Testing
All surviving animals were fasted (18~24 hours) on day 42 for the male test animals, day 56 of the male recovered animals and female satellite animals, and on day 4 of lactation for the female test animals. 5 male test animals in order from smallest animal number, all the male recovery animals and female satellite animals, and 5 female test animals with early parturition dates in order from smallest animal number were selected as measurement subject animals (blood sampling animals).
On the planned day of dissection the day after fasting, animals were anesthetized by intra-abdominal administration of sodium pentobarbital (Nembutal Injection Fluid, Sumitomo Dainippon Pharma Co., Ltd.), and blood drawn from the Caudal Vena Cava. The following items were tested using the sampled blood. Measurements (9) and (10) used 3.2 w/v% trisodium citrate aqueous solution as the anti-coagulation agent, and the plasma obtained through centrifuging at 12,000 rpm (maximum centrifugal acceleration approx. 12,000g) at 4°C, for 3 minutes was used. Measurement of the other items used blood treated with EDTA-2K anticoagulation agent. Remaining samples were discarded after testing completion.
7.10.6 Blood Biochemistry Testing
Some of the blood collected during the planned dissection, after setting for 30 minutes or more at room temperature and shielded from light, was centrifuged at 3,000 rpm (maximum centrifugal acceleration 1,600g) at approximately 4°C for 10 minutes, and the serum obtained used for measurement of the following items. Measurements were made on the day of blood extraction for all females and males of the blood extraction animals. The remaining serum was stored in a freezer at around -80°C (Allowable range: -60°C or lower), and disposed of by the end of the testing.

Sacrifice and pathology:
Organ Weights
Among the planned dissection animals, 5 were selected from each group in order from smallest animal number in the male test animals, all the male recovery animals and female satellite animals, and five with early parturition dates in order from smallest animal number in the female test animals, and the weights measured for the following organs (bilateral organs were measured together). The testes and epididymides are considered accurate indicators of reproductive and developmental toxicity, and therefore these were measured in all cases. The measurements used an electronic scale (AW120L Shimadzu Corporation). In addition, the weights were measured on the day of dissection, and the relative weights (ratio to body weight) were calculated based thereon. Organ weights were not measured for non-pregnant females.
Brain, heart, liver, kidneys, adrenals, thymus, spleen, testes, epididymides

Pathological Dissection Examination
The animals subject to blood testing following blood sampling, and the non-subject animals, had their abdominal aortas severed, and were desanguinated and euthanized, under anaesthesia according to the method of Item 7.10.5, and autopsied. Non-pregnant animals were euthanized by the above method on day 26 after confirmation of mating, and then were likewise autopsied.

Pathohistological Examination
The following organs and tissues were sampled for all animals, fixed in 10vol% neutral phosphate-buffered formalin fluid, and stored.
Brain, pituitary, thymus, lymph nodes (low jaw/mesenteric), trachea, lungs, intestines (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid/parathyroid (bilateral), heat, liver, spleen, kidneys (bilateral), adrenals (bilateral), bladder, testes (bilateral), epididimides (bilateral), seminal vesicles (including congealing gland), prostatic ventral lobe, ovaries (bilateral), uterus, vagina, bone marrow (right femur), sciatic nerve (right), spinal cord, locations of gross abnormalities
In the control group and 1000 mg/kg group, hematoxylin-eosin stain specimens were fabricated by normal methods from the above organs and tissues for 5 animals in order from the smallest animal number in the male test group, and 5 animals with early parturition date in order from the smallest animal number in the female test animals, the locations of gross abnormalities observed in all animals, including the control group, and the ovaries of 2 females (Nos. 50107, 50305) that were non-gravid even though mating had been confirmed, and then microscopically examined. As a result, no further examinations were performed because no changes thought to have been triggered by the tested substance were observed in the 1000 mg/kg group. Moreover, in one female of the 1000 mg/kg group (No. 50409), it was not possible to check the parathyroid on one side of the sample, therefore only the other side of the sample was examined.
Statistics:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All males and females of the 1000 mg/kg group exhibited the drug mixed into faeces (black, colour of tested substance). This was seen in the males and satellite females from day 2 onward, up to day 43, and in females from day 1 before mating onward up to lactation day 5.
In addition, 1 female (No. 50401) in the 1000 mg/kg group, exhibited a tumour on the back on gestation day 18 ~ lactation day 3, and a tumour on the chest on lactation days 4 and 5. This was a mammary adenocarcinoma as a result of pathohistological examination. However, natural occurrences of this change have also been observed, and we determined the change to not have toxicological significance based on the onset conditions.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No changes thought triggered by the tested substance were seen.
In the males of the 1000 mg/kg group, a high body weight tendency was seen from day 29 onward, throughout the administration and recovery periods. However, because this was the opposite change from the low value that would normally be toxicologically problematic, we determined that this change was not toxicologically significant.
In the 40 and 200 mg/kg groups, no significant differences with the control group were seen for both males and females throughout the administration and recovery periods, and trended at an equivalent value.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No changes thought triggered by the tested substance were seen.
High feed intake was seen on days 36, 50, and 54 in the males of the 1000 mg/kg group. However, because this was the opposite change from the low value that would normally be toxicologically problematic, we determined that this change was not toxicologically significant.
In the 40 and 200 mg/kg groups, no significant differences with the control group were seen for both males and females throughout the administration and recovery periods, and they trended at an equivalent value.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes thought triggered by the tested substance were seen.

[At End of Administration Period]
A prolongation tendency in the prothrombin time was seen in the males of the 200 and 1000 mg/kg groups. However, no changes were seen in the other haematological tests, and as a result of having compared the background data (Item 12.6(1)) at the testing facility, and showed that the mean + 2 S.D. of the background data was exceeded, however it was seen in 1, 2 and 1 cases, respectively in the control group, 200, and 1000 mg/kg groups, and we determined from the onset conditions that the change was not toxicologically significant.
In addition, a low lymphocyte ratio and high neutrophil ratio were seen in the leukocyte fractionation of the males of the 200 mg/kg group, however because no dose correlation was seen in any of these, we determined this to be a coincidental change.

[At End of Recovery Period]
A shortened prothrombin time was seen in the females of the 1000 mg/kg group. However, because a similar change was seen in at the end of the administration period, as well as because the change was the opposite of the prolongation that is normally toxicologically problematic, we determined this to be a coincidental change.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
[At Dosing Period End]
Elevated total cholesterol was seen in the males of the 1000 mg/kg group.
In addition, a low ASAT value, or a tendency toward low values, was seen in the males of the 40 mg/kg or higher groups, and a high triglycerides tendency was seen in the males of the 1000 mg/kg group. However, as a result of having compared the background data (Item 12.6(2)) at the testing facility, the mean ASAT (100.8 U/L) of the control group exhibited values in excess of the mean of the background data (93 U/L), while the triglycerides showed a high value in only 1 case (No. 00403), whereas the other 4 cases had values on par with the mean of the background data. We therefore determined that none of these changes were toxicologically significant. In addition, although elevated urea nitrogen was seen in the males of the 200 mg/kg group, no dose-correlation was seen, and therefore we determined this to be a coincidental change.

[At End of Recovery Period]
Elevated glucose and triglycerides were seen in the males of the 1000 mg/kg group. However, because an elevation of the feed intake was seen throughout the recovery period, these changes were all associated with the elevated feed intake, and we determined that the change was not toxicologically significant.
In addition, elevated ALAT and ALP as well as low inorganic phosphorous were seen in males of the 1000 mg/kg group. However, with regard to the elevated ALAT and low inorganic phosphorous, neither of these exhibited similar changes at the end of the administration period, and as a result of having compared these with the background data (Item 12.6(3)) at the testing facility, although 1 animal (No. 00410) exhibited elevated ALAT, because the other 4 had values on par with the background data mean, and the inorganic phosphorous was within the range of the mean ±2 S.D. of the background data in all cases, we determined these were both coincidental changes. In addition, with regard to the ALP elevation, as a result of having compared this with the background data (Item 12.6(3)) at the testing facility, this was due to the mean of the control group (268.0 U/L) being below the mean of the background data (334 U/L).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males :
[During the Administration Period]
In protein, whereas there were 5 animals that exhibited (2+) in the control group and 40 mg/kg group (1 and 3 animals , respectively), significant elevation (2+) was seen in all cases in the 200 and 1000 mg/kg groups. In addition, elevated chlorine was seen in the 1000 mg/kg group.

[During the Recovery Period]
Occult blood (2+) and (3+) respectively exhibited 1 case in the 1000 mg/kg group. However, similar changes were not seen in the administration period, nor were these changes seen in other examinations, therefore we determined these changes to not be toxicologically significant.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Detailed Observation of Symptoms: This was observed in the home cage both during handling as well as in the open field, and no abnormalities were observed in both males and females in any of the groups.
Functional Examination: The responsiveness to stimuli was the same for both males and females of all groups, and no abnormalities were seen. In addition, even when grip strength was measured, no significant differences were seen between the control group and the tested substance administration groups for both males and females.

Measurement of Spontaneous Movement: No changes thought triggered by the tested substance were seen.
A high movement value was seen at 20~30 minutes in the males of the 200 mg/kg group. However no correlation with the dose was seen, and therefore this was determined to be a coincidental change.
No significant differences with the control group were seen for both males and females in the 40 and 1000 mg/kg groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
[At End of Administration Period]
Elevation of the absolute and relative liver weights was seen in the females of the 1000 mg/kg group, and elevation of the relative weights in the females of the 200 mg/kg group. In addition, low relative thymus weights were seen in the females of the 1000 mg/kg group.
In addition, low relative adrenal weights were seen in the males of the 200 and 1000 mg/kg groups. However, as a result of having compared these with the background data (Item 12.6 (5)) at the testing facility, all cases were within the range of the background data mean ±2 S.D., and no associated changes were seen in pathohistological examinations. Therefore we determined that the changes were not toxicologically significant. In addition, although elevated relative kidney weights were seen in the males of the 40 mg/kg, no dose correlation was seen and therefore we determined this to be a coincidental change.

[At End of Recovery Period]
Elevated absolute liver weights and low relative brain and heart weights were seen in the males of the 1000 mg/kg group. However, for the liver, no significant changes were seen in the relative weights, and therefore we determined the changes to not be toxicologically significant. For the heart, similar changes were not seen at the end of the administration period, and as a result of having compared these with the background data (Item 12.6(6)) at the testing facility, all cases were within the range of the background data mean ±S.D., and therefore we determined these to be coincidental changes. As for the brain, an elevation trend was seen in the final weights in the 1000 mg/kg group. We therefore determined this change to be caused by the high body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No changes thought triggered by the tested substance were seen.
In the dissections performed at the end of the administration and recovery periods, several gross changes were seen, however no dose-correlation was seen, and these were found sporadically. Therefore we determined that the changes were not toxicologically significant
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No changes thought triggered by the tested substance were seen.
In the measurement of the organ weights, although low adrenal weights were seen in males, and elevated liver weights and low thymus weights in females, histological changes associated with the weight changes were not seen. In addition, no marked changes were seen in the ovaries of the 2 females where mating was confirmed but were non-gravid. The subdermal tumour found in 1 female (No. 50401) was histologically diagnosed as a mammary adenocarcinoma, but this was determined to be a coincidental change from the onset state.
In addition, various histological changes were seen in each group, including the control group. However, the onset of these was not specific to rats, and no apparent correlation was seen with the dose in the onset conditions. We therefore determined that the changes were not toxicologically significant.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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 absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
Therefore, the no-observed effect level (NOEL) with regard to the repeat dose toxicity under the conditions of this test was 200 mg/kg/day in the males, and 40 mg/kg/day in the females, because elevated total cholesterol was seen in males in the 1000 mg/kg group, and high liver weights for females in the 200 mg/kg group, and the no-observed adverse effect level (NOAEL) to be 1000 mg/kg/day in both males and females because no abnormalities associated with the changes stated above were seen in the pathohistological examination.
Executive summary:

We orally administered copper dibutyldithiocarbamate at doses of 40, 200, and 1000 mg/kg to male and female SD-series rats (Crl:CD(SD)) starting from 14 days prior to mating, and throughout mating, for a total of 42 days for the males, and for the females throughout gestation and parturition up to the 4th day of lactation, and we studied the repeat dosing toxicity and reproductive/developmental toxicity, as well as the recoverability of these changes. The number of animals in a single group was 12 males (including recovered animals), and 12 females (5 animals added to the control group and 1000 mg/kg group as recovered animals), respectively, and in the control group we administered only the medium (0.1% w/v Tween 80-spiked 0.5% w/v CMC-Na aqueous solution).

 

Repeat Dose Toxicity

In blood biochemistry testing, an elevated total cholesterol value was seen in the males of the 1000 mg/kg group. In addition, in the pathology examinations, elevated liver weights were seen in the females of the 200 and 1000 mg/kg groups, however low values were seen for the thymus in the females of the 1000 mg/kg group. Changes associated with these were not seen in the other blood biochemistry testing as well as the pathohistological examinations.

 

In general condition observation, faeces including the drug were observed in both males and females in the 1000 mg/kg group (black, tinted with the tested substance). However, no abnormal content was seen in the intestinal tract during autopsy, and histological abnormalities were not seen in the intestinal tract and other organs/tissues as well. It was therefore determined that these changes were triggered by the colour of the tested substance, and that the changes were not toxicologically significant.

The above changes all disappeared in the 2-week recovery period, and reversibility was seen.

Moreover, no changes triggered by the tested substance were seen in the body weight, feed intake amount, behaviour examination, functional examination, and spontaneous movement measurement, or in the haematological analysis as well as urine analysis.

Therefore, the no-observed effect level (NOEL) with regard to the repeat dose toxicity under the conditions of this test was 200 mg/kg/day in the males, and 40 mg/kg/day in the females, because elevated total cholesterol was seen in males in the 1000 mg/kg group, and high liver weights for females in the 200 mg/kg group, and the no-observed adverse effect level (NOAEL) to be 1000 mg/kg/day in both males and females because no abnormalities associated with the changes stated above were seen in the pathohistological examination.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
It is a reliable study with a klimisch score of 2.

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

Mode of Action Analysis / Human Relevance Framework

28 -day repeated toxicity study (2009):

Copper dibutyldithiocarbamate was administered at doses of 40, 200, and 1000 mg/kg to male and female SD-series rats (Crl:CD(SD)) starting from 14 days prior to mating, and throughout mating, for a total of 42 days for the males, and for the females throughout gestation and parturition up to the 4th day of lactation, and we studied the repeat dosing toxicity and reproductive/developmental toxicity, as well as the recoverability of these changes. The number of animals in a single group was 12 males (including recovered animals), and 12 females (5 animals added to the control group and 1000 mg/kg group as recovered animals), respectively, and in the control group we administered only the medium (0.1% w/v Tween 80-spiked 0.5% w/v CMC-Na aqueous solution).

In blood biochemistry testing, an elevated total cholesterol value was seen in the males of the 1000 mg/kg group. In addition, in the pathology examinations, elevated liver weights were seen in the females of the 200 and 1000 mg/kg groups, however low values were seen for the thymus in the females of the 1000 mg/kg group. Changes associated with these were not seen in the other blood biochemistry testing as well as the pathohistological examinations.

In general condition observation, faeces including the drug were observed in both males and females in the 1000 mg/kg group (black, tinted with the tested substance). However, no abnormal content was seen in the intestinal tract during autopsy, and histological abnormalities were not seen in the intestinal tract and other organs/tissues as well. It was therefore determined that these changes were triggered by the colour of the tested substance, and that the changes were not toxicologically significant.

The above changes all disappeared in the 2-week recovery period, and reversibility was seen.

Moreover, no changes triggered by the tested substance were seen in the body weight, feed intake amount, behaviour examination, functional examination, and spontaneous movement measurement, or in the haematological analysis as well as urine analysis.

Therefore, the no-observed effect level (NOEL) with regard to the repeat dose toxicity under the conditions of this test was 200 mg/kg/day in the males, and 40 mg/kg/day in the females, because elevated total cholesterol was seen in males in the 1000 mg/kg group, and high liver weights for females in the 200 mg/kg group, and the no-observed adverse effect level (NOAEL) to be 1000 mg/kg/day in both males and females because no abnormalities associated with the changes stated above were seen in the pathohistological examination.

Additional information

Justification for classification or non-classification

Proposed self-classification according to the Regulation (EC) No 1272/2008: no classification is required based on the available data.