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Description of key information

Treatment with 1000 mg/kg of the test material was not tolerated. A NOEL (no observed effect level) could not be established as dose-dependent changes in the functional observational battery, hematology, and clinical chemistry were observed in animals of groups 2 (40mg/kg) and/or 3 (200 mg/kg). However, as no histopathological correlates to the clinical pathological functions were found in groups 2 and 3, 200 mg/kg is considered to be the NOAEL (no observed adverse effect level).

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:
13 June - 20 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: HsdCpb:WU
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight in g at study initiation: m: 261 (240 – 285), f: 178 (160 – 194)
- Fasting period before study: no
- Housing: individually under conventional conditions in Type III Makrolon® cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 – 24 °C
- Humidity (%): 49 – 89 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 hours
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): PEG 300 shows low toxicity and is used as standard vehicle in subacute tox studies
- Concentration in vehicle: 0, 8, 40, 200 g/L
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test material suspensions were prepared every 3-4 days. Stability was shown for 5 days.
HPLC analyses performed in week 1 and week 4 of administration revealed a 97-110% of the anticipated concentrations.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
daily (7d/week)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
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:
Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 40 mg/kg bw/day
Male: 10 animals at 200 mg/kg bw/day
Male: 30 animals at 1000 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 40 mg/kg bw/day
Female: 10 animals at 200 mg/kg bw/day
Female: 30 animals at 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No signs of toxicity and no organ alterations at necropsy were detected in rats after single oral administration of 2000 mg/kg body weight

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: 5 male and 5 female rats of groups 1 (control) and 4 (high dose)
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): random
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once on off days

FOB: Yes
- Time schedule:in all animals before the first exposure, and after 1, 2, 3, and 4 weeks of treatment.

MOTOR ACTIVITY: yes
- Time schedule: week 4

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4
- Anaesthetic used for blood collection: Yes (inhalation)
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before the first exposure, and after 1, 2, 3, and 4 weeks of treatment
- Dose groups that were examined: 1-4
- Battery of functions tested:
palpebral closure, ease of removal and handling
from cages, muscle tone, lacrimation, salivation, piloerection, fur appearance, mobility,
arousal, gait, approach response, touch response, click response, tail pinch response,
righting reflex, pupil response, raising, raising behavior, defecation and urination (number
of fecal boluses, feces consistency, number of urine pools, urine stain size), hind limb foot
splay, fore limb and hind limb grip strength, internal body temperature, catalepsies,
posture, convulsions, vocalization, stereotypies and abnormalities

OTHER:
Sacrifice and pathology:
All rats of groups 1-3 surviving until the end of their scheduled treatment period were anesthetized by a carbon dioxide air mixture and exsanguinated by opening the abdominal
vessels. They were subjected to a detailed necropsy and histopathological examination
Statistics:
All parameters were analyzed separately for each sex and time. To take the number of dose groups into account all the test procedures used maintain a multiple significance level of a= 0.05.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
high dose animals showed mortality on days 5-7.
Most symptoms show the general discomfort of the animals in group 4.
Mortality:
mortality observed, treatment-related
Description (incidence):
high dose animals showed mortality on days 5-7
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Generally, the body weight and body weight gain were not impaired by the treatment with the test item up to 200 mg/kg. A slight decrease of body weight gain in the females of groups 2 and 3 was observed, but males in groups 2 and 3 were not affected. Mean body weights of groups 1-3 did hardly differ. Group 4 showed significant decreases of body weight on day 7 (males + females) and day 14 (males). The animals recovered nicely and showed normal body weights on days 21 and 28. Body weight gain had also recovered by day 28.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly decreased on day 7 in group 4 (males and females) by the treatment with the test item in comparison to the control group. Afterwards food consumption returned back to control levels. Groups 2 and 3 did not show any difference from control at all time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption was significantly increased in group 4 on day 7 (males and females) and day 10 (males) compared to the control.
On day 28, a significant increase was observed in group 3 males and females, as well as a significant decrease in group 4 females (days 24 + 28). However, the absolute differences in water consumption were small, and a biological relevance of this finding not confirmed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slight decrease in the number of erythrocytes in group 3 females together with a slight increase in the number of reticulocytes in group 3 males.
Group 4: Hematology revealed a slight decrease in the number of erythrocytes in females together with a slight increase in the number of reticulocytes in males and females at the end of the study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Groups 1-3: Clinical chemistry changes of serum-substrates and proteins together with the changes in serum enzymes indicated a dose-dependent effect on liver and kidney parameters after 4 weeks of treatment with the test item.
Group 4: Changes of the clinico-chemical liver and kidney parameters observed in groups 2 and 3 were not seen in the remaining group 4 animals. As treatment in group 4 animals was stopped during the first week and animals were not treated for approximately 3 weeks, it can be assumed that the changes are reversible.

Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- Statistically significant changes in the 200 mg/kg group: reduced hind limb foot splay on day 7 (females) and day 28 (males) reduced fore limb grip strength on day 28 (males), and reduced hind limb grip strength on day 14 (females) and day 21 (males). Motor activity did not show any significant changes in males and females treated with 40 or 200 mg/kg of the test item at the end of test week 4.
- High dose group: The functional observational battery (FOB) revealed a significantly increased number of animals with piloerection on day 7 in the remaining animals. This finding is probably a sign of general discomfort in these animals. A slight trend was still seen on day 14. Various parameters of the neuromuscular system showed significant changes: hind limb foot splay was decreased on days 7, 14, and 28, mostly in group 4 females. Fore limb grip strength was reduced on days 7, 14, and 28 in some mid and high dose groups. Hind limb grip strength was reduced on days 7, 14, 21, and 28 in various treatment groups of both genders. Additionally, the righting reflex was decreased in group 4 animals on day 7.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group 3: increased absolute and relative liver weights, increased absolute and relative thyroid and adrenal weights, increased absolute and relative adrenal weights
Group 4: No evaluation of group 4 organ weights was feasible due to the reduced number of animals that survived the study until day 29.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy revealed in animals that died or were killed moribund in the high dose the following treatment-related findings: Enlarged adrenals in 2 females. In the kidney a mottled discoloration and enlargement were found in 3 males. Discolorations of the liver were found in 4 females, one female showed an additional enlargement of the liver. A small spleen was found in 3 males and 2 females. In the stomach dark/red discolorations were found in 5 males and in 5 females. One female showed minimal focal ulcerations. A small thymus was found in 5 males 3 females. In the urinary bladder a red content was found in 5 males and 3 females. In high dose animals that survived until day 29 one female showed a unilateral granular surface of the kidney and 2 females showed discolorations in the stomach.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Group 3 and 2 rats showed no treatment-related histopathological findings.
- Group 4: cortical hyperplasia in the adrenals, atrophy of the bone marrow, vacuolar degeneration of cortical tubules in the kidney with formation of tubular casts and dilation of Bowman’s space of the glomerula, hepatocellular hypertrophy with focal necroses in the liver, lymphoid depletion in the lymph nodes, the spleen and the thymus, focal mucosal ersions/ulcerations in the stomach, single cell necroses in the parotid gland and reduced zymogen in the exocrine pancreas. These findings were present in animals that died/killed moribund and to a minor degree in single animals that survived until day 29.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
clinical biochemistry
clinical signs
haematology
mortality
Key result
Critical effects observed:
no

Treatment with 1000 mg/kg of test item was not tolerated. A NOEL could not be etablished as dose-dependent changes in the functional observational battery, hematology, and clinical chemistry were observed in animals of groups 2 (40mg/kg) and/or 3 (200mg/kg). However, as no histopathological correlates to the clinical pathological functions were found in groups 2 and 3, 200 mg/kg is considered to be the NOAEL.

Conclusions:
200 mg/kg is considered to be the NOAEL (no observed adverse effect level).
Executive summary:

Study design


The test material was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of male and female HsdCpb:WU Wistar rats at doses of 40, 200, or 1000 mg/kg. A similarly constituted control group received the vehicle, polyethylene glycol 300 (PEG 300), and served to generate contemporary control data.
The control and high dose groups consisted of 10 male and 10 female rats each. The low and mid dose groups consisted of 5 male and 5 female rats each. At the end of the treatment period 10 (5 males and 5 females) rats were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period. The rats were kept under conventional conditions.

Daily dose levels and number of rats used:

Group Dose / [mg/kg] Animal Nos. Planned main kill Planned recovery kill
Males Females Males Females Males Females
1 0 1-10 31-40 1-5 31-35 6-10 36-40
2 40 11-15 41-45 11-15 41-45
3 200 16-20 46-50 16-20 46-50
4 1000 21-30 51-60 21-25 51-55 26-30 56-60



Treatment of high dose rats had to be terminated within the first week of dosing since 7/10 males and 5/10 females were either found dead or had to be killed for humane reasons. All surviving rats including group 1 control animals were killed at the end of the 4 week treatment period.

Group Dose / [mg/kg] Necropsy after 4 weeks
Males Females
1 0 1-10 31-40
2 40 11-15 41-45
3 200 16-20 46-50
4 1000 26, 28, 29 52-55, 58


Survey of inlife investigations:

Observations/Measurements Time schedule
Appearance and behavior daily
Mortality daily
Motor activity week 4
Functional observational battery predose, weeks 1, 2, 3, and 4
Body weight once a week
Food consumption once a week
Water consumption twice a week
Hematology week 4
Clinical chemistry week 4
Urinalysis week 4


Results

The high dose animals showed mortality on days 5-7, therefore treatment was stopped within 4-7 days. All remaining animals of the high dose group as well as animals groups 1-3 were kept until the scheduled main kill. For the sake of completeness, the results of group 4 animals are described. However, only animals of groups 1-3 received treatment throughout the whole treatment period of 28 days.

Groups 1-3:

The functional observational battery revealed some statistically significant changes in the 200 mg/kg group: reduced hind limb foot splay on day 7 (females) and day 28 (males) reduced fore limb grip strength on day 28 (males), and reduced hind limb grip strength on day 14 (females) and day 21 (males).

Motor activity did not show any significant changes in males and females treated with 40 or 200 mg/kg at the end of test week 4.

Body weight, body weight gain, food, and water consumption were not impaired by the treatment at doses of up to 200 mg/kg.

Hematology after 4 weeks of treatment revealed a slight decrease in the number of erythrocytes in group 3 females together with a slight increase in the number of reticulocytes in group 3 males.

Clinical chemistry changes of serum-substrates and proteins together with the changes in serum enzymes indicated a dose-dependent effect on liver and kidney parameters after 4 weeks of treatment.

Specific urinary gravity and urinalysis did not reveal any toxicologically relevant changes.

Determination of organ weights revealed increased absolute and relative liver weights for group 3 males and females. A tendency to increased adrenal weights was found for group 3 males and females. Group 3 and 2 rats showed no treatment-related histopathological findings. No histopathological correlated of the hematological or clinico-chemical changes were found.

Group 4:

The high dose animals showed mortality on days 5-7, therefore treatment was stopped within 4-7 days. All remaining animals of the high dose group were kept until the scheduled main kill.

The functional observational battery (FOB) revealed a significantly increased number of animals with piloerection on day 7 in the remaining animals. This finding is probably a sign of general discomfort in these animals. A slight trend was still seen on day 14. Various parameters of the neuromuscular system showed significant changes: hind limb foot splay was decreased on days 7, 14, and 28, mostly in group 4 females. Fore limb grip strength was reduced on days 7, 14, and 28 in some mid and high dose groups. Hind limb grip strength was reduced on days 7, 14, 21, and 28 in various treatment groups of both genders. Additionally, the righting reflex was decreased in group 4 animals on day 7.

The motor activity could only be evaluated in females (because the scheduled males had died prematurely). They did not show any changes from control.

Body weight and body weight gain were significantly impaired on days 7 and 14 (except females on day 14).

Hematology revealed a slight decrease in the number of erythrocytes in females together with a slight increase in the number of reticulocytes in males and females at the end of the study.

Changes of the clinico-chemical liver and kidney parameters observed in groups 2 and 3 were not seen in the remaining group 4 animals. As treatment in group 4 animals was stopped during the first treatment week and animals were not treated for approximately 3 weeks, it can assumed that the changes are reversible.

Specific urinary gravity and urinalysis did not reveal any toxicologically relevant changes. At necropsy the following main treatment-related changes were found in high dose animals: enlarged adrenals, discolorations in kidney, liver, stomach, red-colored urine, enlargement of the liver, small spleen and thymus and ulcerations in the stomach. No evaluation of group 4 organ weights was feasible due to the reduced number of animals that survived the study until day 29.

Histopathological examinations of animals that died or were killed moribund revealed the following main treatment-related changes in high dose animals:

  • minimal to moderate diffuse cortical hyperplasia in the adrenals
  • mild atrophy or single cell necroses in the bone marrow
  • mild to massive vacuolar degeneration of cortical tubules in the kidney with formation of tubular casts and dilation of Bowman’s space in the glomerula
  • minimal to mild hepatocellular hypertrophy with minimal to moderate focal necroses, minimal to massive deposition of fat in hepatocytes (females) in the liver
  • moderate to severe lymphoid depletion in the lymph nodes, the spleen and the thymus
  • minimal to mild focal mucosal ersions and focal ulceration in the stomach
  • single cell necroses in the parotid gland
  • reduced zymogen in the exocrine pancreas.


Single high dose animals that survived until day 29 revealed the following findings:

  • mild deposition of fat in cortical tubules of the kidney and basophilic tubules
  • minimal hepatocellular hypertrophy with minimal necroses
  • massive lymphoid depletion in the spleen
  • mild depletion in the thymus.
  • mild diffuse cytoplasmic vacuolation of zona fasciculata cells in the adrenals.

Overall, the findings in animals that survived show qualitative similarities to those observed in animals that died but with a decreased incidence and severity.

Conclusion

Treatment with 1000 mg/kg of the test material was not tolerated. A NOEL (no observed effect level) could not be established as dose-dependent changes in the functional observational battery, hematology, and clinical chemistry were observed in animals of groups 2 (40mg/kg) and/or 3 (200 mg/kg). However, as no histopathological correlates to the clinical pathological functions were found in groups 2 and 3, 200 mg/kg is considered to be the NOAEL (no observed adverse effect level).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD Guideline study under GLP conditions

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

Justification for classification or non-classification

The result of the OECD 407 guideline study show that the test item does not meet the criteria for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

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