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Administrative data

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422), rat: NOAEL ≥ 1000 mg/kg bw/day

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
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Feb - 27 Mar 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, presentation of data in written and graphical format is limited. Based on the available data, the NOAEL derived by the study director was changed from 300 mg/kg bw/day to 1000 mg/kg bw/day.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bureau For Chemical Substances, Poland
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University, Białystok, Poland
- Age at study initiation: approximately 11 weeks old
- Weight at study initiation: males: 346.2-367.1 g, females: 210.2-217.8 g
- Housing: The animals were kept in plastic cages covered with wire bar lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). During the experiment, there were 3 or 5 animals/cage. Each sex was kept separately. For the purpose of mating, one female and one male were placed together. Pregnant females were housed individually. After delivery, females were housed with their offspring.
- Diet: Murigran standard granulated laboratory fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz, Poland; ad libitum
- Water: tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 35-48
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item were prepared daily (directly before the administration).

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle: 0.5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of the dose level of the test item were conducted by BLIRT S.A. Laboratory, Trzy Lipy 3/1.38, 80-172 Gdańsk, Poland.
Samples (aqueous solutions of the dose levels) collected on each treatment day were frozen (ca. - 20°C) and sent to BLIRT S.A. (143 samples in four batches). The samples stored in a freezer (at -20±5 °C) were stable for at least 25 days.
The dosel levels in the samples were within the following ranges:
18.815 - 20.533 mg/mL for the dose level of 20 mg/mL (94.1 – 102.7%)
59.104 - 62.994 mg/mL for the dose level of 60 mg/mL (98.5 – 105.0 %)
199.265 - 220.722 mg/mL for the dose level of 200 mg/mL (99.6 – 110.4%).
The results were within the range of 80 -120%.
Duration of treatment / exposure:
males: 28 days
females: 43-49 days (2 weeks prior to mating; variable time to conception; duration of pregnancy; until 4 days after delivery)
satellite groups (males and females): 28 days (observation for 14 days post-treatment)
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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:
10 males and 12 females per dose
5 males and 5 females per satellite group (vehicle satellite control group and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the outcome of a 7-day dose range finding study, the dose levels of 100, 300, and 1000 mg/kg bw/day were selected for the main study.
- Post-exposure recovery period in satellite groups: 14 days
Two satellite groups of five animals per sex in the control and the high dose group were used for observation of reversibility, persistence or delayed occurrence of systemic toxic effects. The satellite groups were treated for 28 days and observed for 14 days post-treatment. The animals of the satellite groups were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (once a day on weekends and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day (once a day on weekends and public holidays)

BODY WEIGHT: Yes
- Time schedule for examinations: males: twice per week; females: twice per week during pre-mating; on Days 0, 7, 14 and 20 of gestation; on Day 0 or Day 1 post-partum and on Day 4 post-partum
Body weights of pups were measured on Day 0 or Day 1 post-partum and on Day 4 post-partum.

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (the animals were anesthetised with a xylazine-ketamine mixture at the dose of 10 mg xylazine/kg bw and 100 mg ketamine/kg bw to collect blood samples from the heart)
- Animals fasted: Yes (for about 18 h)
- How many animals: 5 males and 5 females from each dose group
- The following parameters were examined: numbers of leukocytes, erythrocytes, and thrombocytes; the concentration of hemoglobin, hematocrit, MCV (mean cell volume - erythrocyte), MCH (Mean Corpuscular Hemoglobin), and MCHC (Mean Corpuscular Hemoglobin Concentration); prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (the animals were anesthetised with a xylazine-ketamine mixture at the dose of 10 mg xylazine/kg bw and 100 mg ketamine/kg bw to collect blood samples from the heart)
- Animals fasted: Yes (for about 18 h)
- How many animals: 5 males and 5 females from each dose group
- The following parameters were examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT); alkaline phosphatase (AP); total protein, albumin, total cholesterol, urea nitrogen, creatinine, glucose, bilirubin, bile acids, sodium, potassium, chlorides and calcium
The globulin level was counted by subtracting albumin from total protein; the ratio of albumin to globulin was calculated (A/G ratio)

URINALYSIS: Yes
- Time schedule for collection of urine: for 18 h during the last day of the experiment
- Metabolism cages used for collection of urine: Yes (for about 18 h)
- How many animals: 5 males and 5 females from each dose group
- The following parameters were examined: general urinalysis: urine colour and volume, specific gravity (density), pH, proteins, ketones, bilirubin, blood, urobilinogen, leukocytes, and glucose; urine sediment: leukocytes, erythrocytes, bacteria, and crystals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period (1-2 days before euthanasia)
- Dose groups that were examined: all dose groups (5 males and 5 females from each dose group)
- Battery of functions tested: sensory activity / grip strength / motor activity

BONE MARROW EXAMINATIONS:
- Time schedule for collection: after euthanasia bone marrow was collected from the dissected femurs
- The following parameters were examined: erythrocyte system: proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatic erythroblasts; leukocyte system: myeloblasts, promyelocytes, orthochromatophilic and acidophilic myelocytes, orthochromatophilic and acidophilic metamyelocytes, rod neutrophils and rod eosinophils, filamented neutrophils, filamented eosinophils, and basophils; different cells: lymphocytes, monocytes, plasmocytes, megakaryocytes, and, other cells, (cells of reticulum, mast cells, and bare nuclei)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content
weights of internal organs: testes, epididymides and ovaries (of all surviving animals); brain, thymus, heart, liver, spleen, kidneys, and adrenal glands of 5 adult males and 5 adult females from each group
HISTOPATHOLOGY: Yes
The following organs and tissues were examined: brain with the cerebellum, spinal cord, muscle with the peripheral nerve, salivary glands, lymph nodes, trachea, thyroid with the parathyroids, stomach, duodenum, jejunum, ileum, caecum, colon, liver, pancreas, spleen, lungs, heart, thymus, kidneys, adrenal glands, urinary bladder, ovaries, uterus with the cervix, testicles, epididymides, accessory sex glands (prostate with the seminal vesicles and coagulating glands), and organs showing gross lesions
Statistics:
Student’s t-test (p ≤ 0.05); Dunnett’s test (p ≤ 0.05)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two males from the low dose group and two females from the high dose group died during the treatment period (not treatment-related)
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males from the low dose group and two females from the high dose group died during the treatment period (not treatment-related)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in the average body weight in high dose females on gestation Day 20 (non-adverse)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant differences observed for MCV, MCH, erythrocytes and reticulocytes in the high dose groups (non-adverse).
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Slight decrease or increase in arousal in all dose groups; reduced latency of pain responses; reduced forelimb grip strength of high dose males; (incidental)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Thymus and spleen weights (absolute and relative) from high dose males were statistically significantly changed; in high dose females spleen weights were statistically significantly increased regarding absolute and relative organ weights (non-adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Perforation of the esophagus caused by gavage error in two males of the low dose group and one high dose female (not related to the test substance)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lesions were observed in several tissues; however, none of the lesions observed had a specific character or showed severity with increasing dose level (non-adverse)
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two males from the low dose group (Day 25 and Day 28) and two females from the high dose group (Day 5 and Day 39) died during the treatment period. In both males and in the female that died on Day 39 perforation of the esophagus was found, indicating a gavage error. These deaths were not considered to be treatment-related. In the second female that died no abnormalities were found at necropsy and the death was considered to be incidental.
Decreased locomotor activity, a decreased rate of reaction to sound, and bleeding from the snout were observed in one male in the low dose group (the animal was found dead on Day 28). Alopecia and a scab on the snout were noted in one male from the mid dose group (temporary changes). There were no other abnormal differences in appearance and behaviour between the treated and the control groups. Animals in the satellite groups showed no delayed occurrence of adverse effects.

BODY WEIGHT AND WEIGHT GAIN
During the pre-mating and post-mating period, there were no statistically significant differences in average male body weights between the treated and the control groups (see Table 1).
In females of the high dose group, the average body weight was statistically significantly decreased on gestation day 20 compared to the control group (see Table 3). However, the body weights were found to be comparable with control group in the lactation phase (see Table 4). As body weights of the high dose females were only decreased by 8.1% (control females: 340.1 ± 22.91 g; high dose females (1000 mg/kg bw/day): 312.7 ± 27.44 g), this finding was not considered to be toxicologically relevant. According to Chou et al. (1998), body weight loss or decrease in body weight gain of less than 10% is considered non-adverse. In females of the other dose groups, there were no statistically significant differences in average body weights between the treated and the control groups during the pre-mating, gestation and lactation period (see Tables 2-4). Animals in the satellite group showed no statistically significant differences in body weights of males and females.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmic examinations did not reveal any pathological changes.

HAEMATOLOGY
A statistically significant decrease in the number of erythrocytes and a statistically significant increase in MCV and MCH levels were observed in males and females in the high dose group compared with the control group (see Tables 5 and 6). Furthermore, the number of reticulocytes in males from the high dose group was statistically significantly increased. No other hematological parameters showed any significant differences between the treated and the control groups.
Animals in the satellite group showed only a statistically significant increase in MCV in females.
Considering various historical control data for hematological parameters for Wistar (CRL:WI) rats (published by Charles River Laboratories) the data indicate that the statistically significant differences observed for MCV, MCH, erythrocytes and reticulocytes in the high dose group are within or similar to the normal biological variation of Wistar rats.
In addition, the differences in hematological parameters for the Wistar rats are within or similar to the range of historical control values of CD® rats and F-344 rats (Derelanko, 2008).
Therefore, it can be considered that the differences in hematological parameters in male and female Wistar rats, although statistically significant, are unlikely to be treatment-related effects and thus non-adverse.
The examination of the bone marrow erythrocyte system showed an increase in the numbers of polychromatophilic and orthochromatic erythroblasts as well as an increase in the total number of cells in high dose males. In high dose males also a decrease in the total number of cells in the leukocyte system and a decrease in the leukocyte system/erythrocyte system quantitative ratio was observed. No other statistically significant changes in the bone marrow erythrocyte/leukocyte system in treated animals compared with the controls were found. Animals in the satellite group showed no statistically significant changes in the bone marrow erythrocyte/leukocyte system and in the numbers of different cells in bone marrow in the satellite group. Therefore, the observed effects were not considered to be treatment-related.

CLINICAL CHEMISTRY
No treatment-related findings in biochemical and enzymatic determinations were observed. The only statistically significant effects were a decreased albumin concentration and a decreased albumin/globulin ratio in males from the mid dose group. As these effects were not observed in the high dose group, there was no dose-response relations ship, the effects are not considered to be toxicologically relevant.
Animals in a satellite group showed no persistence of the observed effects or delayed occurrence of toxic effects regarding biochemical or enzymatic parameters.

URINALYSIS
No significant changes were observed in the urinalysis parameters assessed in all the dosed animals. The only finding was an increase in the number of leukocytes in females from the high dose group. The urine of all animals was yellow. Animals in a satellite group showed no delayed occurrence or persistence of effects. Thus, the effect is not considered to be toxicologically relevant.
The results of the urine sediment examinations in all dose groups showed no statistically significant changes. The only finding was a decrease in the number of crystals in males from mid dose group. Animals in a satellite group showed no delayed occurrence or persistence of effects.
As no dose-response relationship was noted and the effect was not observed in the satellite group, the finding was considered to be non-adverse.

NEUROBEHAVIOUR
Open field observations
Males and females from all dose groups showed no changes in involuntary clonic and tonic movements, changes in gait, or atypical behaviour compared with the control animals.
A slight decrease in arousal was noticed in one female in the low dose group, in three females in the mid dose group and in one male and one female in the high dose group. A slight increase in arousal was noticed in three males and two females in the low dose group, in one male in the mid dose group and in one male in the high dose group.
There were no statistically significant differences in the number of fecal boluses in the dose groups compared with the control group. A statistically significant decrease in the number of urine pools in high dose males was observed. All other treatment groups were not affected.
No statistically significant differences in horizontal and vertical locomotor activities of treated males and females were observed compared with the control group.
Animals in the satellite group showed only a slight decrease in arousal in one male in the satellite control group and a slight increase in arousal in two females in the satellite control group, and in one male and one female in the satellite group. In the post-treatement period, a slight decrease in arousal was observed in one male and two females in the satellite control group and in two males and two females in the satellite group. The observed effects were considered to be incidental due to the occurrence of decreased/increased arousal in the control groups and therefore not toxicologically relevant.

Evaluation of sensorimotor responses to stimuli
There were no treatment-related effects on responses to visual, tactile, auditory, and pain stimuli. The results of the latency of pain responses measurements of males and females showed that there were no differences between the treated and the control groups.
In animals in the satellite group, the latency period in males was significantly shorter than in the respective satellite control group. No other effects were observed in the animals from the satellite group. The latency of pain responses in males is considered to be incidental.

Measurement of locomotor activity
The forelimb grip strength of high dose males was significantly weaker, compared with the control animals. Males in the low and mid dose groups and females in all treatment groups were not affected. No statistically significant differences in the hindlimb grip strength were observed in any dose group.
There were no statistically significant differences in the fore- and hindlimb grip strength of males and females in the satellite group, indicating that the observed effect on forelimb strength in high dose males was incidental. In the post-treatment observation, the forelimb grip strength of males from the satellite group was significantly greater than that of the satellite control group. No effects were observed for other treated males and females.

ORGAN WEIGHTS
The analysis of absolute and relative weights of internal organs of the animals from the high dose group showed statistically significant changes compared with the control group. A decrease in absolute and relative weights of the thymus and an increase in absolute and relative weights of the spleen in high dose males were found (see Table 7). In high dose females, an increase in absolute and relative weights of the spleen, and in relative weights of the kidneys were observed (see Table 8). In males and females from the low and mid dose groups, no statistically significant changes were noted compared to the control group. The analysis of absolute and relative weights of the gonads showed a statistically significant increase in absolute weights of the ovaries in females in the low dose group (see Table 10).
Males in the satellite group showed statistically significantly decreased thymus weights (absolute) and statistically significantly increased epididymides weights (relative) (see Table 9). In females in the satellite group a statistically significant increase in absolute and relative weights of the kidneys and a statistically significant decrease in relative weights of the brain were observed. No other statistically significant changes in absolute and relative weights of the remaining organs were noted in the satellite group.
No macroscopic or microscopic abnormalities were found in these organs during the gross pathology and histopathological examinations. Furthermore, the toxicokinetic behaviour of the test substance gives no indication that any of the organs with changed weights are target organs of the test substance. According to the Ecetoc technical report No. 85 (2002), increased organs weights with no evidence of pathological and histopathological changes should be considered to be a non-specific response (i.e. not adverse).
It is common understanding that alterations in organ weights are not necessarily toxicologically relevant if there are no other indications of organ-specific adverse effect and should therefore not be considered as an adverse effect when no macroscopic abnormalities and no histopathological findings were observed for in the organs. Thus, the observed effects were not considered to be treatment-related.

GROSS PATHOLOGY
Macroscopic changes of parental animals that died during the study period:
In the low dose group two males died during the study period. In both males perforation of the esophagus was observed indicating gavage error. The presence of clear fluid in the pleural cavity of the lungs and swelling of the right part of the body, i.e. the cervical and the scapular parts were found.
In the high dose group two females died during the study period. In one female esophageal perforation and the presence of clear fluid in the thoracic cavity was noted. In the second female no abnormalities were found.
Macroscopic changes of parental animals euthanized at the end of the treatment period:
In the control group, an inflammatory focus around the left ventricle of the heart in one male and paleness of the liver and a lesion in the left liver lobe in one female were observed. There were no other gross changes in any other dose group. There were no macroscopic changes in the organs and tissues of the rats in the satellite group.

HISTOPATHOLOGY:
The histopathological examination of the parental animals that died during the treatment period revealed lesions associated with perforation of the esophagus and damage to surrounding tissues (circulatory disorder- hyperemia of the salivary glands, mandibular lymph nodes, thymus, lungs, and heart, as well as emphysema in the lungs), indicating a gavage error.
The histopathological examination of organs and tissues of the euthanized parental animals at the end of the study revealed a few changes in individual animals. Lesions were observed in the brain, cerebellum, lungs, heart, liver, spleen, pancreas, intestines (duodenum, jejunum, cecum, and colon), kidneys, adrenals, and prostate. The histopathological changes observed in these organs were associated with various types of disorders. However, none of the lesions observed in the treated animals had a specific character or showed severity with increasing dose levels. Most of them occurred in the control and the treated groups at the same time with the same incidence. Some of them were observed only in the control group.
The histopathological examination of organs and tissues of the euthanized animals in the satellite group revealed a few changes in individual animals. Lesions were observed in the cerebellum, heart, liver, adrenals and prostate. The histopathological changes observed in these organs were associated with various types of disorders. However, none of the lesions observed in the treated animals had a specific character or showed severity with increasing dose level.
Therefore, the observed effects were not considered to be treatment-related.

References:
ECETOC Technical Report 85, 2002, Recognition of, and differentiation between, adverse and non-adverse effects in toxicological studies, ISSN-0773-6347-85
Chou et al., 1998, Minimal risk level (MRLs) for hazardous substances. J. Clean Technol., Environ. Toxicol., & Occup. Med., Vol. 7, No. 1, 1998
Derelanko, M.J., 2008, The toxicologist´s pocket handbook. 2nd edition, informa healthcare, ISBN 987-142005138-4
Charles River Laboratories, 2008, Clinical laboratory parameters for Crl:WI (Han)
Charles River Laboratories, 1998, Baseline hematology and clinical chemistry values for Charles River Wistar rats – (Crl:WI(BR)) as a function of sex and age. Technical bulletin
Charles River Technical data (available online at www.criver.com)
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:
other: NOAEL corresponding to the highest dose level tested
Critical effects observed:
not specified

Table 1: Average body weights [g]: pre-mating and after mating periods – parental males

 

Week

Group [mg/kg bw/day]

0

n=10

100

n=10

300

n=10

1000

n=10

0

367.1

±

25.80

367.0

±

29.81

367.0

±

25.98

367.1

±

22.74

1

386.9

±

28.00

385.1

±

32.26

384.4

±

28.66

375.8

±

26.62

2

400.6

±

28.90

399.2

±

32.61

394.1

±

31.77

384.9

±

23.24

3

406.6

±

31.90

402.7

±

33.48

400.3

±

33.42

392.7

±

25.83

4

415.2

±

34.00

402.3

±

36.72

410.2

±

36.02

404.0

±

27.98

n -number of test animals; mean ± SD

 

Table 2: Average body weights [g]: pre-mating and after mating periods – parental females

 

Week

Group [mg/kg bw/day]

0

100

300

1000

n=12

n=12

n=12

n=12/11*

0

217.6

±

16.08

217.8

±

11.94

217.8

±

17.46

217.7

±

18.78

1

228.3

±

16.35

225.6

±

12.58

226.6

±

16.46

227.4

±

16.75

2

233.3

±

16.38

231.8

±

11.98

233.0

±

16.08

236.9

±

17.04

n -number of test animals; *-one female died on day 5 of the experiment; mean ± SD

 

Table 3: Average body weights [g]: gestation period – parental females

 

Day

Group [mg/kg bw/day]

0

n=12

100

n=12

300

n=12

1000

n=11#

0

231.5

±

14.37

231.3

±

11.87

231.1

±

16.40

235.8

±

15.24

7

252.0

±

17.23

247.1

±

14.60

250.0

±

19.16

250.5

±

20.37

14

279.0

±

20.54

271.8

±

16.54

275.5

±

23.37

270.3

±

23.30

20

340.1

±

22.91

328.9

±

21.87

333.3

±

33.41

   312.7   ±   27.44*

n -number of test animals; #included not pregnant female; *statistically significant difference at p ≤ 0.05: mean ± SD

Table 4: Average body weights [g]: lactation period – parental females

 

Day

Group [mg/kg bw/day]

0n=12

100n=12

300n=12

1000

n=9

0

262.4

±

19.59

253.2

±

15.84

261.6

±

25.53

251.2

±

22.43

4

269.2

±

24.52

262.8

±

20.31

264.6

±

24.48

260.1

±

22.54

n -number of test animals; mean ± SD

Table 5: Hematological examinations in males

 

Parameter

GROUP [mg/kg bw/day]

0

n=5

100

n=5

300

n=5

1000

n=5

 

HEMOGLOBIN

 

g/L

 

158.60±7.67

 

155.60±1.95

 

159.00±5.92

 

150.80±4.02

 

HEMATOCRIT

 

1/1

 

0.47 ±0.02

 

0.46 ±0.01

 

0.46 ±0.03

 

0.45 ±0.01

 

ERYTHROCYTES

 

x1E12/L

 

8.90 ±0.41

 

9.14 ±0.57

 

8.91 ±0.59

 

7.81 ±0.19*

 

MCV

 

fL

 

52.40±1.14

 

50.80±1.79

 

51.60±1.14

 

57.20±1.92*

 

MCH

 

pg

 

17.82±0.65

 

17.08±0.86

 

17.90±0.86

 

19.34±0.66*

 

MCHC

 

g/L

 

341.40±4.93

 

337.40±6.11

 

346.20±11.34

 

337.80±5.45

 

RETICULOCYTES

 

1/1

 

0.020±0.001

 

0.019±0.006

 

0.018±0.004

 

0.037±0.006*

 

THROMBOCYTES

 

x1E9/L

 

576.80±48.96

 

590.60±45.38

 

554.60±82.70

 

534.20±30.56

 

LEUKOCYTES

 

x1E9/L

 

3.26 ±0.76

 

4.06 ±2.05

 

2.62 ±0.27

 

2.98 ±0.73

 

PT

 

s

 

14.38±1.04

 

13.34±1.52

 

13.52±1.25

 

13.16±0.84

 

APTT

 

s

 

47.40±3.05

 

44.40±5.32

 

42.80±5.50

 

43.20±4.15

n -number of test animals; *statistically significant difference at p ≤ 0.05: mean ± SD

Table 6: Hematological examinations in females

 

Parameter

GROUP [mg/kg bw/day]

0

n=5

100

n=5

300

n=5

1000

n=5

 

HEMOGLOBIN

 

g/L

 

148.40±10.92

 

138.20±6.46

 

145.40±9.81

 

135.40±5.46

 

HEMATOCRIT

 

1/1

 

0.44 ±0.02

 

0.40 ±0.03

 

0.43 ±0.02

 

0.40 ±0.02

 

ERYTHROCYTES

 

x1E12/L

 

7.87 ±0.63

 

7.42 ±0.47

 

7.61 ±0.54

 

6.74 ±0.45*

 

MCV

 

fL

 

55.60±1.82

 

53.20±1.30

 

56.00±1.22

 

59.60±1.67*

 

MCH

 

pg

 

18.86±0.61

 

18.64±0.65

 

19.10±0.46

 

20.12±0.67*

 

MCHC

 

g/L

 

340.00±6.36

 

349.40±17.40

 

340.20±5.76

 

338.40±3.97

 

RETICULOCYTES

 

1/1

 

0.038±0.016

 

0.038±0.013

 

0.046±0.013

 

0.060±0.015

 

THROMBOCYTES

 

x1E9/L

 

796.60±159.91

 

803.60±165.02

 

790.40±52.78

 

662.60±52.11

 

LEUKOCYTES

 

x1E9/L

 

4.70 ±1.15

 

4.98 ±1.48

 

4.78 ±0.59

 

4.54 ±1.18

 

PT

 

s

 

11.90±0.46

 

11.68±0.49

 

11.78±0.39

 

11.32±0.56

 

APTT

 

s

 

49.40±1.34

 

45.80±4.44

 

56.40±18.82

 

44.80±6.18

n -number of test animals; *statistically significant difference at p≤ 0.05: mean ± SD

 

 

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:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 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

The combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD guideline 422 and in compliance with GLP (Przybyła, 2015).

The test substance was orally administered via gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Male rats (10/dose group) were dosed for at least 28 days and female rats (12/dose group) for 43-49 days (14 days prior to mating, throughout pregnancy and up to and including Day 4 post-partum). An additional satellite group of five animals per sex in the control and the high dose group was included to assess the reversibility, persistence or delayed occurrence of systemic toxic effects, for at least 14 days post- treatment. Animals of the satellite groups were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.

During the study period two males from the low dose group (Day 25 and Day 28) and two females from the high dose group (Day 5 and Day 39) died. At necropsy in both dead males and in one female perforation of the esophagus was noted, indicating a gavage error. In the second female no abnormalities were found at necropsy. These deaths were not considered to be treatment-related.

Decreased locomotor activity, a decreased rate of reaction to sound, and bleeding from the snout was observed in one male in the low dose group (the animal was found dead on day 28). Alopecia and a scab on the snout were noted in one male from the mid dose group (temporary changes). There were no other abnormal differences in appearance and behaviour between the treated and the control groups. The animals in the satellite group showed no delayed occurrence of adverse effects.

During the pre-mating and post-mating period, there were no significant differences in average male body weights between the treated and the control groups.

In females of the high dose group, the average body weight was significantly decreased on gestation Day 20, compared with the control group. However, the body weights were found to be comparable to the control group in the lactation phase. As body weights of the high dose females were only decreased by 8.1%, this finding was not considered to be toxicologically relevant. According to Chou et al. (1998), body weight loss or decrease in body weight gain of less than 10% is considered to be non-adverse.

In females of the low and mid dose groups, there were no significant differences in average body weights between the treated and the control groups during the pre-mating, gestation and lactation period. Animals in the satellite group showed no significant changes in body weights of males and females.

A significant decrease in the number of erythrocytes and a significant increase in MCV and MCH were observed in males and females in the high dose group, compared with the control group. Furthermore, the number of reticulocytes in males from the high dose group was significantly increased. No other hematological parameters showed any significant changes between the treated and the control groups.

Animals in the satellite group showed only a significant increase in MCV in females.

Historical control data for hematological parameters for Wistar (CRL:WI) rats (published by Charles River Laboratories) indicate that the significant differences observed for MCV, MCH, erythrocytes and reticulocytes in the high dose group are within or similar to the normal biological variation of Wistar rats.

The differences in hematological parameters for the Wistar rats are within or similar to the range of historical control values of CD® rats and F-344 rats (Derelanko, 2008).

Therefore, the differences in hematological parameters in male and female Wistar rats are considered to be non-adverse.

The examination of the bone marrow erythrocyte system showed a significant increase in the numbers of polychromatophilic and orthochromatic erythroblasts as well as an increase in the total number of cells in high dose males. In high dose males a decrease in the total number of cells in the leukocyte system and a decrease in the leukocyte system/erythrocyte system quantitative ratio was observed. No other significant changes in the bone marrow erythrocyte/leukocyte system in treated animals compared with the controls were found. Animals in the satellite group showed no significant changes in the bone marrow erythrocyte/leukocyte system and in the numbers of different cells in bone marrow.

No treatment-related findings in biochemical and enzymatic determinations were observed. Also urinalysis determinations showed no significant changes of all dose groups compared to the control group. In animals of the satellite group no abnormalities regarding biochemical, enzymatic or urinalysis parameters were found.

Males and females from all dose groups showed no involuntary clonic and tonic movements, changes in gait, or stereotypical behaviour compared to the control animals.

There were no treatment-related effects on responses to visual, tactile, auditory, and pain stimuli. The forelimb grip strength of high dose males was significantly weaker compared with the control animals. The males in the low and mid dose groups and all treated females were not affected. No significant differences in the hindlimb grip strength were observed in any dose groups.

Animals in the satellite group showed no significant differences in the fore- and hindlimb grip strength, indicating that the observed effect in high dose males was incidental. In the 14-day post-treatment observation period after treatment, the forelimb grip strength of males from the satellite group was significantly greater compared with satellite control group. No effects were observed in other treated males and females.

A decrease in absolute and relative weights of the thymus and an increase in absolute and relative weights of the spleen was observed in high dose males. In high dose females, an increase in absolute and relative weights of the spleen, and relative weights of the kidneys were observed compared with the control group. In males and females from the low and mid dose groups, no significant changes in organ weights were noted. There were no other significant changes in absolute and relative weights of the remaining organs in any other dose group.

Males in the satellite group showed significantly decreased thymus weights (absolute) and significantly increased epididymides weights (relative). In females in the satellite group a significant increase in absolute and relative weights of the kidneys and a statistically significant decrease in relative weights of the brain were observed. No other significant changes in absolute and relative weights of the remaining organs were noted in the satellite group.

No macroscopic or microscopic abnormalities were found when investigating these organs. Furthermore, the toxicokinetic behaviour of the test substance gives no indication that any of the organs with changed weights are target organs of the test substance. According to the ECETOC technical report No. 85 (2002), increased organs weights with no evidence of pathological and histopathological changes should be considered as non-specific response (i.e. not adverse).

It is common understanding that alterations in organ weights are not toxicologically relevant and should therefore not be considered as an adverse effect when no macroscopic abnormalities and no histopathological findings were observed for these organs. Thus, the observed effects were not considered to be treatment-related.

Gross pathology results of the males that died during the study period showed perforation of the esophagus indicating gavage error. The presence of clear fluid in the pleural cavity of the lungs and swelling of the right part of the body, i.e. the cervical and the scapular parts were found. In the high dose group two females died during the study period. In one female esophageal perforation and the presence of clear fluid in the thoracic cavity was noted. In the other female no abnormalities were found.

No treatment-related macroscopic effects were observed in any main group or satellite group during necropsy.

The histopathological examination of the parental animals which died during the treatment period revealed lesions associated with perforation of the esophagus and damage to surrounding tissues (for example wastage of the wall of the esophagus, circulatory disorder- hyperemia of the salivary glands, mandibular lymph nodes, thymus, lungs, and heart, as well as emphysema in the lungs), indicating a gavage error.

No treatment-related microscopic effects were observed during the histopathological examination in any main group or satellite group. The few lesions noted were considered to be incidental as no dose-related increase in number or severity was noted, and the lesions were observed in all groups.

In conclusion, the treatment of male and female rats with the test substance at the dose levels of 100, 300 and 1000 mg/kg bw/day by oral gavage revealed no adverse effects. Based on the results of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL for systemic toxicity was considered to be ≥ 1000 mg/kg bw/day.

Justification for classification or non-classification

The available data on repeated dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.