Registration Dossier

Administrative data

Description of key information

OECD 422 (oral, rat): NOAEL = 50 mg/kg bw/day for systemic toxicity and local effects in stomach

Key value for chemical safety assessment

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:
24 Nov 2016 - 21 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST (Han Wistar) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (RMS) UK Limited
- Age at study initiation: Females: 98 - 104 days; Males: 84 - 90 days
- Weight at study initiation: Females: 192 - 232 g; Males: 323 - 362 g
- Housing: Up to 5 animals/sex were housed during pre-pairing, up to 5 males after mating. During gestation and lactation, one female (+ litter) was housed per cage. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. Grid bottomed polypropylene cages were used during pairing, when one male and one female were co-housed. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified pelleted (GLP Certificated rat and mouse breeding diet), ad libitum
- Water: Potable water, ad libitum
- Acclimation period: Females: 20 days; Males: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Nov 2016 To: 21 Jul 2017
Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose in water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The required amount of test material was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of test material in test formulations were analysed gravimetrically for the study, confirming accurate formulation. Homogeneity was confirmed gravimetrically. The mean analysed concentration for the three samples (5, 13.5 and 35 mg/mL) remained within 10% of the mean value and the variation was less than 10%.
Duration of treatment / exposure:
Males: 5 weeks; Females: Two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation
Frequency of treatment:
Once daily, 7 days/week
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
equivalent to: 45.2 mg/kg bw/day as active ingredient
Dose / conc.:
135 mg/kg bw/day (nominal)
Remarks:
equivalent to: 122.1 mg/kg bw/day as active ingredient
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
equivalent to: 316.6 mg/kg bw/day as active ingredient
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a 2-week preliminary toxicity study in the Han Wistar rat conducted at the same testing facility. In that study, dose levels of 50, 125 and 300 mg/kg bw/day were well tolerated with no clinical signs or abnormal macroscopic findings. There was a marked initial reduction in male body weight gain at 300 mg/kg bw/day and an overall marked reduction of weight gain over Days 1-15 of study in females receiving 300 mg/kg bw/day, compared with animals receiving 50 mg/kg bw/day. As the LD50 has been reported as 500 mg/kg (tesed at the same testing facility), a high dose level of 350 mg/kg bw/day (as active ingredient) was selected for use on this study. The low dose remained at 50 mg/kg bw/day. The geometric mean was 132.3 mg/kg bw/day, so 135 mg/kg bw/day was selected as the mid dose level to allow for assessment of a dose response.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, animals were inspected visually and at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).

DETAILED CLINICAL OBSERVATIONS: Yes, detailed observations, detailed physical examination and arena observations were performed in parental animals.
- Time schedule: Males were inspected daily in Week 1 and thereafter once each week. Females were inspected daily in Week 1, once in Week 2, on Days 0, 7, 14 and 20 during gestation phase and on Days 1, 6 and 12 during lactation phase. Sensory reactivity and grip strength assessments were performed and motor activity were measured (all before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.

BODY WEIGHT: Yes, the body weight of all animals was recorded
- Time schedule for examinations: For males, before dosing on the day of treatment and weekly thereafter, and on the day prior to necropsy and on the day of necropsy. For females, before dosing on the day that treatment commenced (Day 1) and weekly before pairing, on Day 0, 7, 14 and 20 after mating, on Day 1, 4, 7 and 13 of lactation and on the da prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded.
- the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: The five lowest numbered surviving males and the first five females with a surviving litter, in each dose group.
- Parameters checked: Haematocrit, haemoglobin concentration, erythrocyte count, absolute reticulocyte count, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, red cell distribution width, total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, basophils, monocytes, large unstained cells), plateled count, prothrombin time, activated partial thromplastin time, T4 hormone

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes. overnight
- How many animals: The five lowest numbered surviving males and females per group.
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, bile acids, urea, creatinine, glucose, total cholesterol, tryglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin and albumin/globulin ratio
Sacrifice and pathology:
GROSS PATHOLOGY: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Males were sacrificed after Week 5 investigations were completed. Females were sacrificed either on Day 25 after mating, if the females failed to produce a viable litter, or on or after the day the last offspring died, if the litter died before Day 13, or on Day 14 of lactation following terminal blood sampling.
Organs checked: Adrenals, bone marrow smear, brain (including cerebrum, cerebellum and pons), cecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum , jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), lymph nodes (left axillary and mesenteric) , ovaries, Peyer’s Patch, pituitary, prostate, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus (including cervix and oviducts) and vagina

HISTOPATHOLOGY: Histopathological examination was performed for 5 surviving males and 5 lactating femals with surviving litter each of the control and of the highest dose group, respectively, and for all animals in case of abnormalities occurred. Dehydrated tissue samlples were embebbed in paraffin wax
Organs checked: Adrenals, brain (including cerebrum, cerebellum and pons), cecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum , jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), lymph nodes (left axillary and mesenteric) , ovaries, Peyer’s Patch, prostate, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus (including cervix and oviducts) and vagina
Statistics:
For grip strength, motor activity, clinical pathology, litter size and survival indices, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Similarly, for the litter average ano-genital distance, analysis of covariance was performed using the average pup body weight for each litter as the covariate.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Sporadical salivation and/or post dose salivation was observed in 6/10 males and in 6/10 females of the mid dose group, and in 8/10 males and all females of the highest dose group, respectively. In 1/10 high-dosed males, piloerection and underactivity were observed after the first dose administration, which disappeared thereafter.
At the routine weekly examination, vocalisation was observed in 3/10 females receiving 50 mg/kg bw/day, 2/10 males and 3/10 females receiving 135 mg/kg bw/day, and 5/10 males and 1/10 females receiving 350 mg/kg bw/day when compared with 1/10 control males and 3/10 control females.
The only other clinical sign observed was ventral body hair loss in one female receiving 135 mg/kg/day during lactation; this was not considered to be related to treatment with the test substance according to the study director.

According to the study director, salivation and or post dose salivation staining was observed sporadically throughout the treatment period, these signs are all likely to relate to the dosing process and taste of the formulation rather than to systemic toxicity. Vocalisation is also believed to be due to an aversion to the dosing process.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduced bodyweight gain was observed during Days 1 - 8 (62% lower), 8 - 15 (37% lower), 15 - 22 (92% lower), 1 - 15 (54% lower) and 1 - 36 (43% lower) of treatment for males receiving 350 mg/kg bw/day, when compared with controls. In addition, the mean body weight was significantly lower in the high-dose groups compared with the control groups. While food consumption in males given 350 mg/kg bw/day was lower during the first two weeks of treatment, it was not comparably different from controls during the remaining duration of the study so the depressed body weights cannot entirely be related to a reduction in feeding.

Group mean bodyweight and bodyweight change for females receiving the test substance was similar to controls prior to pairing, however bodyweight change was statistically significantly lower during Days 0 - 7 (27% lower) and 7 - 14 (30% lower) of gestation for females receiving 350 mg/kg bw/day when compared with controls. Bodyweight gain was lower (75% lower) during Days 1 - 4 of lactation for females receiving 350 mg/kg bw/day. During Days 7 - 13 of lactation bodyweight gain was statistically significantly higher for females receiving 350 mg/kg bw/day when compared with controls. However, the group mean values were within the same range as the control values, therefore the effect on body weight in females was not considered to be toxicologically relevant.

Please refer to Table 1 and Table 2 under "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption was low for males receiving 350 mg/kg bw/day during Week 1 of treatment (20% lower) and to a lesser extent in Week 2 (12% lower) when compared to controls.
At 350 mg/kg bw/day food intake of females was marginally low during Week 1 of treatment (15% lower) and marginally but statistically significantly low during gestation (11% lower during Days 7-12 and 10% lower during Days 13-19) and was 17% lower (but not statistically significant) during Days 1 to 3 of lactation. Food consumption was similar to controls for females receiving 50 or 135 mg/kg bw/day of the test substance prior to pairing, during gestation and lactation.

Please refer to Table 3 under "any other information on results incl. tables".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination at termination, following five weeks of treatment for males and seven weeks for females revealed statistically slightly lower male haematocrit levels and lower female prothrombin time for all treatment groups when compared with controls.
Haemoglobin levels were statistically lower and lymphocyte counts were statistically higher for males receiving 135 or 350 mg/kg bw/day when compared with controls. Mean cell haemoglobin, mean cell volume and activated partial thromboplastin time were all statistically reduced and white blood cell, neutrophil and eosinophil counts were statistically higher for males receiving 350 mg/kg bw/day when compared with controls.

According to the study director, the observed haematological findings at termination were considered to be of unclear toxicological significance since these findings had no supporting macroscopic or microscopic treatment related changes detected. The smaller size of the circulating erythrocytes in these animals appears to be responsible for the lower hematocrit and hemoglobin levels as red blood cell counts were similar for treated and control animals. The reduced hemoglobin levels were only marginally reduced to levels that are not considered to adversely affect the health of the animals. The higher white blood cell counts recorded in males receiving 350 mg/kg bw/day, and to a much lesser extent at 135 mg/kg bw/day, may be linked to the inflammatory cell infiltrate in the stomach.

Please refer to Table 4 and Table 5 under "any other information on results incl. tables".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with the controls, glucose (79 and 78%, respectively) and creatinine (88 and 83%, respectively) levels were decreased for males receiving 135 or 350 mg/kg bw/day. Potassium (111 and 113%, respectively) and phosphorus (118 and 131%, respectively) levels were statistically significantly higher for males receiving 135 mg/kg bw/day or 350 mg/kg bw/day; phosphorus (126%) levels were also statistically significantly higher for females receiving 350 mg/kg bw/day.
Chloride (98 and 94% for males and females, respectively) levels were statistically significantly lower for animals receiving 350 mg/kg bw/day and total protein (90%) and albumin (92%) levels were statistically significantly lower for males receiving 350 mg/kg bw/day.
Plasma levels of bile acids for males and females treated the test substance were highly variable, and statistical significance was not attained, these findings were considered due to individual variation.
All other inter-group differences from controls were minor, were confined to one sex or lacked a dose-relationship; these values were considered fortuitous and were therefore considered of no toxicological importance.

Please refer to Table 4 and Table 5 under "any other information on results incl. tables".
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Group mean hindlimb grip strength values for females receiving 350 mg/kg bw/day were statistically significantly low when compared with controls.
The majority of group mean activity scores for both high and low beams for all groups of treated males and females were low compared with controls, and statistical significance was achieved for males receiving 135 mg/kg bw/day at the 42-minute interval and the total score and for males receiving 350 mg/kg bw/day at the 6, 24 and 42-minute interval and the total score (all high beam). The low beam total score for females receiving 350 mg/kg bw/day has attained statistical significance as has the 18 and 42-minute interval and total score for females receiving 350 mg/kg bw/day (also low beam). There was a clear dose-related reduction at both high and low beam activity as seen by the total scores for both males and females.

According to the study director, the aetiology of the dose related low activity scores for total high and low beams is uncertain as there were no corresponding clinical observations indicating reduced activity. The cause of the low group mean hindlimb grip strength values for females receiving 350 mg/kg bw/day remains unclear as there were no clinical macroscopic or microscopic findings which could be clearly linked to this finding.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weights for males killed after 5 weeks of treatment revealed significantly lower terminal bodyweight and the group mean unadjusted (78%) and adjusted (70%) thymus weights were lower for males that received 350 mg/kg bw/day, when compared with controls although the difference was not statistically significant. Adjusted kidney weights (112%) were marginally but statistically higher in high-dosed males than in controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dark areas were seen in the glandular mucosa of the stomach of animals treated at all dose levels and in controls:
Control: 0/10 males and 2/10 females
50 mg/kg bw/day: 1/10 males and 1/10 females
135 mg/kg bw/day: 0/10 males and 3/10 females
350 mg/kg bw/day: 5/10 males and 1/10 females

According to the study director, In the glandular stomach, inflammatory cells, predominantly neutrophils, were scattered throughout the mucosa frequently in association with eosinophilic globules and in some cases with apoptosis of the mucous neck cells. A dose response was apparent and both incidence and severity were generally higher in male animals. Mucosal congestion was seen at minimal severity in two control females, only associated with a minimal, focal, infiltrate of inflammatory cells in one of them, whereas in treated animals, it was either more severe or associated with a constellation of findings distinct from that seen in the controls. Mucosal erosion was present at a low incidence but at all dose levels in females and in one male given 350 mg/kg bw/day. The constellation of changes seen in the stomach is indicative of an irritation effect of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach
Inflammatory cells, predominantly neutrophils, were scattered throughout the glandular mucosa in both sexes, all dose levels and one control female, mostly at minimal severity. In most males and three females given 350 mg/kg bw/day, this was accompanied by minimal to moderate eosinophilic globules in mucous neck cells. This same change was seen at minimal severity in four males given 135 mg/kg bw/day and one male given 50 mg/kg bw/day. These two changes were occasionally associated with a minimal degree of apoptosis of the mucous neck cells. A submucosal inflammatory cell infiltrate, mostly lymphocytic, was present in five males and one female given 350 mg/kg bw/day, and in one male given 50 mg/kg bw/day. Mucosal congestion was seen in four males given 350 mg/kg bw/day, in two females given 135 mg/kg bw/day and in two control females, and correlated in most cases with dark areas seen at necropsy. Minimal to slight mucosal erosion was seen at low incidence in males given 350 mg/kg bw/day and females at all dose levels. Overall, the changes in the stomach were seen at a higher incidence and severity in males.

Thymus
Involution/atrophy, which can be background change, was seen at minimal severity but higher incidence in males given 350 mg/kg bw/day and correlated with decreased absolute and adjusted thymus weight.

Prostate
Mixed inflammatory cell infiltrate was seen in the dorsolateral lobe in most males given 350 mg/kg bw/day at minimal to moderate severity and at minimal to slight severity in six males given 135 mg/kg bw/day.

Epididymides
Increase in luminal cell debris was seen at minimal to slight severity in eight males given 350 mg/kg bw/day and at minimal severity in three males given 135 mg/kg bw/day, one male given 50 mg/kg bw/day and one control.

According to the study director, increased cell debris, mostly located in the epididymal head and at minimal severity, was seen in eight males given 350 mg/kg bw/day and three males given 135 mg/kg bw/day. A multifocal, minimal to moderate, infiltrate of mixed inflammatory cells was seen in the dorsolateral prostate at the same dose levels. A dose response in incidence and severity was apparent.

Please refer to Table 6 under "any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 45.2 mg/kg bw/day as active ingredient
Sex:
male/female
Basis for effect level:
haematology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
350 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Body weight and body weight change - group mean values (g) for males (F0)

Dose level Day Day Day Day Day Day Change Change Change Change Change
(mg/kg/day) 1 8 15 22 29 36 1-8 8-15 15-22 22-29 29-36
Control 338 356 372 384 401 412 19 16 12 17 11
10.3 10.9 12.9 14.1 15.5 13.8 6.1 5.0 7.3 3.5 3.5
10 10 10 10 10 10 10 10 10 10 10
50 342 359 377 390 408 420 17 18 14 18 12
9.4 11.1 11.7 16.7 18.8 20.6 5.9 5.5 9.5 5.1 4.6
10 10 10 10 10 10 10 10 10 10 10
135 341 358 374 387 405 417 17 17 13 18 12
12.3 17.1 19.2 20.0 18.9 18.3 6.9 4.2 6.5 6.9 7.5
10 10 10 10 10 10 10 10 10 10 10
350 338 344* 354** 355** 373** 379** 7** 10* 1* 17 6
6.8 9.2 12.5 21.6 22.7 24.8 4.3 5.5 11.4 6.8 4.9
10 10 10 10 10 10 10 10 10 10 10

Table 2: Body weight and body weight change - group mean values (g) for females (F0)

Gestation Period
Dose level (mg/kg bw/day)   Day 0 Day 7 Day 14 Day 20 Change 0 - 7 Change 7 - 14 Change 14 - 20 Change 0 - 20
Control Mean 221 243 266 323 22 23 57 102
SD 11.6 10.8 9.9 13.2 3.7 5.3 7.2 9.8
N 9 9 9 9 9 9 9 9
50 Mean 224 246 267 329 21 21 62 105
SD 10.1 14.3 18.6 27.4 5.8 5.2 12.2 18.4
N 10 10 10 10 10 10 10 10
135 Mean 226 246 269 323 20 22 54 97
SD 6.7 8.2 7.9 10.4 5.2 2.6 5.9 6.4
N 10 10 10 10 10 10 10 10
350 Mean 225 241 257 314 16* 16** 57 89
SD 11.3 8.2 9.6 18.8 5.7 5.0 10.5 16.2
N 9 9 9 9 9 9 9 9
Lactation Period
Dose level (mg/kg bw/day)   Day 1 Day 4 Day 7 Day 13 Change 1 - 4 Change 4 - 7 Change 7 - 13 Change 1 - 13
Control Mean 253 256 268 280 4 12 12 27
SD 11.1 13.2 16.7 19.6 7.3 7.3 11.8 16.7
N 9 9 9 9 9 9 9 9
50 Mean 255 260 265 284 5 5 19 29
SD 14.0 19.0 20.1 19.0 8.7 7.9 7.6 10.8
N 10 10 10 10 10 10 10 10
135 Mean 257 260 269 285 3 9 15 27
SD 10.5 11.8 13.4 12.5 5.9 6.3 8.1 9.2
N 10 10 10 10 10 10 10 10
350 Mean 245 246 256 280 1 10 25** 35
SD 7.8 10.7 10.2 11.1 7.3 8.4 6.4 8.5
N 9 9 8 8 9 8 8 8

Table 3: Food consumption - group mean values (g/animal/day) for females during gestation (F0)

Control Mean 18 19 21
SD 0.8 1.6 1.7
N 9 9 9
50 Mean 18 19 21
SD 2.2 1.8 2.1
N 10 10 10
135 Mean 17 20 21
SD 1.3 1.4 1.8
N 10 10 10
350 Mean 16* 17* 19*
SD 1.0 0.9 1.4
N 9 9 9

Table 4: Hematology and clinical chemistry in males

Dose level (mg/kg bw/day)   Hct Hb MCH MCV WBC N L E APTT Creat Gluc
  L/L g/dL pg fL x109/L x109/L x109/L x109/L sec µmol/L mmol/L
Control Mean 0.464 16.0 19.0 55.1 3.65 1.01 2.51 0.06 16.0 35 8.15
SD 0.0105 0.55 0.92 1.56 0.573 0.111 0.589 0.015 1.35 2.6 0.806
N 5 5 5 5 5 5 5 5 5 5 5
50 Mean 0.444* 15.4 18.5 53.2 4.20 0.96 3.09 0.07 17.5 33 7.60
SD 0.0073 0.26 0.14 0.26 1.492 0.236 1.320 0.015 2.00 2.3 1.184
N 5 5 5 5 5 5 5 5 5 5 5
135 Mean 0.447* 15.3* 18.2 53.0* 4.86 0.87 3.81* 0.09 16.4 31* 6.48**
SD 0.0129 0.43 0.63 2.04 0.714 0.228 0.548 0.036 1.35 2.7 0.543
N 5 5 5 5 5 5 5 5 5 5 5
350 Mean 0.443* 14.9** 17.3** 51.7** 5.51* 1.34* 3.92* 0.13** 12.8* 29** 6.35**
SD 0.0181 0.44 0.55 1.61 1.110 0.270 0.898 0.025 2.54 3.9 0.432
N 5 5 5 5 5 5 5 5 5 5 5

Hct: Hematocrit

Hb: Hemoglobin concentration

MCH: Mean cell hemoglobin

MCV: Mean cell volume

WBC: Total leucocyte count

N: Neutrophils

L: Leucocytes

E: Eosinophils

APTT: Activated partial thromboplastin time

Creat: Creatinin

Gluc: Glucose

Table 5: Hematology and clinical chemistry in females

Dose level (mg/kg bw/day)   PT Cl Phos
  sec mmol/L mmol/L
Control Mean 27.4 96 2.39
SD 1.85 3.0 0.493
N 4 5 5
50 Mean 23.5* 94 2.57
SD 2.10 3.0 0.325
N 5 5 5
135 Mean 24.1* 95 2.61
SD 2.52 4.4 0.283
N 5 5 5
350 Mean 22.5** 90* 3.02*
SD 0.33 2.1 0.381
N 5 5 5

PT: Prothrombin time

Cl: Chloride

Phos: Inorganic phosphorus

Table 6: Summary of histopathological findings in males and females

Group/sex Males Females
Dose (mg/kg bw/day) 0 50 135 350 0 50 135 350
Summary of findings in the glandular stomach
Mucosal inflammatory cell infiltrate                
Minimal 0 2 5 6 1 1 3 4
Slight 0 1 0 0 0 0 0 0
Total 0 3 5 6 1 1 3 4
Eosinophilic globules, mucous neck cells                
Minimal 0 1 4 3 0 0 0 2
Slight 0 0 0 2 0 0 0 0
Moderate 0 0 0 2 0 0 0 1
Total 0 1 4 7 0 0 0 3
Apoptosis, mucous neck cells                
Minimal 0 1 1 5 0 0 1 3
Total 0 1 1 5 0 0 1 3
Submucosal inflammatory cell infiltrate                
Minimal 0 1 0 2 0 0 0 1
Slight 0 0 0 3 0 0 0 0
Total 0 1 0 5 0 0 0 1
Mucosal congestion                
Minimal 0 0 0 3 2 0 1 0
Slight 0 0 0 1 0 0 1 0
Total 0 0 0 4 2 0 2 0
Mucosal erosion                
Minimal 0 0 0 0 0 2 2 0
Slight 0 0 0 1 0 0 0 1
Total 0 0 0 1 0 2 2 1
Number of tissues examined 5 5 5 7 6 6 5 6
Summary of findings in the thymus
Involution/atrophy                
Minimal 0 0 0 3 0 0 0 0
Slight 0 0 0 0 1 0 0 0
Total 0 0 0 3 1 0 0 0
Number of tissues examined 5 5 5 5 5 0 0 6
Summary of findings in the prostate
Mixed inflammatory cell infiltrate, dorsolateral lobe                
Minimal 0 0 4 4 - - - -
Slight 0 0 2 2 - - - -
Moderate 0 0 0 1 - - - -
Total 0 0 6 7 - - - -
Number of tissues examined 10 10 10 10 - - - -
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfill the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD 422 and under GLP conditions, 10 male and 10 female Wistar rats received Zinc, bis[O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S]bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) by gavage at dose levels of 50, 135 and 350 mg/kg bw/day (equivalent to 45.2, 122.1 and 316.6 mg/kg bw/day active ingredient); dose selection was based on the results of a preliminary range-finding study at the same testing facility (Envigo CSR, 2017). Males were exposed for 5 weeks, and females were exposed for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Parental animals were observed at least twice daily for mortality and clinical signs. Body weights and food consumption were recorded weekly. All adult animals were subject to a detailed necropsy. At necropsy, haematological, clinical biochemistry parameters and organ weights were determined. Histopathological examination was performed for 5 surviving males and 5 lactating females with surviving litter each of the control and of the highest dose group, respectively, and for all animals in case of abnormalities occurred. Mortality and toxicologically relevant clinical signs were not observed up to the end of the study period. Significant reduced body weight gain was observed in high-dose males when compared with controls. While food consumption in high-dose males was lower during the first two weeks of treatment, it was not comparably different from controls during the remaining duration of the study; the depressed body weights cannot entirely be related to a reduction in feeding. Body weight change was significantly lower during the whole gestation period and during Days 1 – 4 of lactation in high-dosed females, when compared with controls. However, the body weight group mean values were within the same range as the control values, therefore the effect on body weight in females was not considered to be toxicologically relevant. The observed haematological findings at termination were considered to be of unclear toxicological significance since these findings had no supporting macroscopic or microscopic treatment-related changes detected. The smaller size of the circulating erythrocytes in these animals appears to be responsible for the lower hematocrit and hemoglobin levels as red blood cell counts were similar for treated and control animals. The reduced hemoglobin levels were only marginally reduced to levels that are not considered to adversely affect the health of the animals. The higher white blood cell counts recorded in males receiving 350 mg/kg bw/day, and to a much lesser extent at 135 mg/kg bw/day, may be linked to the inflammatory cell infiltrate in the stomach. Regarding the behaviour, the aetiology of the dose related low activity scores for total high and low beams is uncertain as there were no corresponding clinical observations indicating reduced activity. The cause of the low group mean hindlimb grip strength values for females receiving 350 mg/kg bw/day remains unclear as there were no clinical macroscopic or microscopic findings which could be clearly linked to this finding. At gross necropsy, treatment-related changes were seen in the stomach in both sexes, in the thymus in males, as well as in the prostate and epididymides. In the glandular stomach, inflammatory cells, predominantly neutrophils, were scattered throughout the mucosa frequently in association with eosinophilic globules and in some cases with apoptosis of the mucous neck cells. A dose response was apparent and both incidence and severity were generally higher in male animals. Mucosal erosion was present at a low incidence but at all dose levels in females and in one male given 350 mg/kg bw/day. The constellation of changes seen in the stomach is indicative of an irritation effect of the test substance. According to the study director, the higher white blood cell counts recorded in males receiving 350 mg/kg bw/day, and to a much lesser extent at 135 mg/kg bw/day, may be linked to the inflammatory cell infiltrate in the stomach. Increased cell debris, mostly located in the epididymal head and at minimal severity, was seen in eight males given 350 mg/kg bw/day and three males given 135 mg/kg bw/day. A multifocal, minimal to moderate, infiltrate of mixed inflammatory cells was seen in the dorsolateral prostate at the same dose levels. A dose response in incidence and severity was apparent.

Based on haematological effects seen at the lowest test dose, which however were only minimal and not adverse, the NOAEL with respect to systemic toxicity was set at 50 mg/kg bw/day(45.2 mg/kg bw/day as active ingredient).

Justification for classification or non-classification

The available data from an oral OECD 422 study with the registered substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.