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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(2008)
Deviations:
yes
Remarks:
No recovery was considered, only two doses used instead of three as recommended by guideline OECD 407.
Principles of method if other than guideline:
The study was based around the OECD guideline 407, which however, was not fully applicable as this study was intended for dose selection for a follow up 90-day study in rats. No recovery was considered; only two doses were used instead of three as recommended by guideline. The dose level of 250 mg/kg bw/day was established as LOAEL. Animals of this group were initially treated with 500 mg/kg bw/day. However, since severe toxicity was seen, treatment was stopped after 9 days, followed by a 5 day recovery period. Subsequently, dosing of these animals was decreased to 250 mg/kg bw/d. Therefore, it could not be excluded that the initial dosing with 500 mg/kg bw/day had, to some degree, contributed to the overall toxicity on which the LOAEL was based.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid (yellow powder)
- Analytical purity: 99.9 %
- Lot/batch No.: 37973FC4AA
- Expiration date of the lot/batch: 2010-12-01
- Stability under test conditions: stable, solutions of test article are sensitive to light
- Storage condition of test material: At room temperature protected from sunlight

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males), 9 weeks (females)
- Weight at study initiation: 288 - 293 (males), 174 - 180 (females)
- Housing: in groups of 5 per sex in Macrolon cages (MIV type)
- Diet ad libitum: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH)
- Water ad libitum: tap water
- Acclimatisation: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 21.7
- Humidity (%): 37 - 72 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
The choice of vehicle was based on trial formulations performed at NOTOX and on information from the sponsor.
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were kept away from light as much as possible using amber coloured glassware and/or tin foil. Correction was made for specific gravity of the vehicle.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analysis of stability, homogeneity and concentration of the test article under test conditions was not performed as part of the present study. According to the author, such analyses were to be done within a one-generation reproduction toxicity study performed later.
Duration of treatment / exposure:
28 days

Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Reduced to 250 mg/kg bw/day after 9 treatment days a 5 recovery days.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
The test substance, formulated in vehicle, was administered daily for 28 days by oral gavage to the rats. After 9 days and because of severe toxicity, treatment with the highest dose level of 500 mg/kg bw/day (group 3) was stopped for a period of five days (recovery); Subsequently, the same animals were dosed with 250 mg/kg bw/day for 28 days.

Examinations

Observations and examinations performed and frequency:
MORTALITY AND CLINICAL OBSERVATIONS
The animals were checked for mortality at least twice daily and were examined at least daily for clinical signs.

BODY WEIGHT
Weekly, and additionally on day 11; terminal body weight was also assessed.

FOOD CONSUMPTION
Weekly

HAEMATOLOGY AND CLINICAL CHEMISTRY
Blood samples were collected under iso-flurane anaesthesia on the day of sacrifice after a over night fasting. For details on parameters see table 1.

Sacrifice and pathology:
GROSS PATHOLOGY
All animals were anaesthetized at test ending using iso-flurane and subsequently exsanguinated. After gross pathology the following tissues and organs were collected from all animals and fixed: Lung, adrenal glands, spleen, epididymides, sternum with bone marrow, kidneys, testes, liver and all gross lesions.
From all males sperm samples were taken from the proximal part of the vas deferens to assess sperm motility.

Organ weights: Adrenal glands, liver, kidneys, spleen

HISTOPATHOLOGY
Organ and tissue samples were processed, embedded, cut and stained for histopathology.
All tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group, the samples of the testes of groups 1 and 3 (examination of spermatogenesis) and all gross lesions were examined.
Based on treatment-related morphologic changes in the liver, kidneys, adrenal glands, and sternum with bone marrow of group 3 males and/or females, these organs were also examined from all animals of Group 2 (males and females; kidneys were examined for males only).
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
- The percentage of motile spermatozoa and progressive motile spermatozoa were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference.
If the results of the ANOVA were significant (p < 0.05), the Wilcoxon test was applied to the data to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.


Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs consisted of hunched posture, piloerection, a lean appearance, salivation, retching and/or gasping among animals at 500 mg/kg bw/day. These symptoms resolved during the wash-out period of 5 days. During the subsequent treatment at 250 mg/kg bw/day, no toxicologically relevant clinical signs were observed.
One case of rales was seen for 1 female at 250 mg/kg bw/day. This is commonly noted in rats of this age and strain under the conditions in this study.
Salivation was noted intermittently among most animals at 250 mg/kg bw/day as well as among most females at 100 mg/kg bw/day. This effect was considered to be of non-toxicological relevance. No clinical signs were noted among control animals and males at 100 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight loss (up to 14% of the weight recorded on Day 1) or reduced weight gain was noted among all animals at 500 mg/kg bw/day (see figure). At commencement of treatment at 250 mg/kg/day, body weights of females were similar to control values, whilst body weights of males were still slightly lower than control levels (achieving level of statistical significance). During the first two weeks of treatment at 250 mg/kg bw/day, body weight gain of this group was similar to control levels (taking Day 15 as reference point). Although body weight gain in the last two weeks of treatment could not be compared to concurrent control levels (control animals were sacrificed after 28 days), body weight gain was in the range to be expected for rats of this age and strain.
A statistically significant higher body weight gain of females at 100 mg/kg bw/day on day 15 remained within the range considered normal for rats of this age and strain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A lower (relative) food consumption was noted for males and females at 500 mg/kg bw/day, which recovered to control levels after the recovery period.
For females at 250 mg/kg bw/day, relative food consumption appeared slightly higher than control levels. This was due to slightly higher food consumption, as was also observed for females at 100 mg/kg bw/day. Since food consumption levels for these animals remained within the range considered normal, this was considered to be of no toxicological relevance.
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):
The following changes in hematology parameters were of statistical significance in comparison to control animals:
Higher prothrombin time (PT) in males at 100 and 250 mg/kg bw/day, lower reticulocyte and platelet counts in males at 250 mg/kg bw/day, lower hemoglobin concentration and activated partial thromboplastin time (APTT) in females at 250 mg/kg bw/day. However, the both latter effects were within the range considered normal for rats of this age and strain.
Other hematological parameters of animals at 100 and 250 mg/kg bw/day were similar to control values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following in clinical biochemistry parameters were of statistical significance in comparison to control animals:
Higher alanine aminotransferase activity in males at 250 mg/kg bw/day, and in females at 100 and 250 mg/kg bw/day (not statistically significant at 250 mg/kg bw/day), higher inorganic phosphate levels in females at 250 mg/kg bw/d, lower total protein levels in males at 250 mg/kg bw/day, lower urea levels in males at 250 mg/kg/day, lower sodium levels in males at 250 mg/kg bw/day, lower chloride levels in males and females at 250 mg/kg bw/day. However, the four latter effects were within the range considered normal for rats of this age and strain.
Other clinical biochemistry parameters of animals at 100 and 250 mg/kg/day were similar to control values.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver weight and liver to body weight ratio was higher in males and females at 100 and 250 mg/kg bw/day. A lower spleen weight was recorded for females at 250 mg/kg bw/day, while spleen to body weight ratio was lower for females at 100 and 250 mg/kg bw/day. Kidney and adrenal weights of animals at 100 and 250 mg/kg bw/day were similar to control levels.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At macroscopic examination greenish discolouration of the kidneys was observed among all animals at 250 mg/kg bw/day. Redbrown discolouration of the liver was noted among most females at 250 mg/kg bw/day.
Other necropsy findings included dark-red foci on the pancreas, enlarged adrenal glands, tan discolouration of adrenal glands, and pelvic dilation of the kidneys. These changes were not accompanied by treatment-related histopathological correlates, and hence these were considered not to be of toxicological relevance.
No abnormalities were noted at necropsy of control animals and females at 100 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following histopathological changes were considered to be related to treatment with the test substance:
Liver: Hypertrophy of the hepatocytes in 4/5 males and 5/5 females of group 3.
Kidneys (males): increased incidence and severity of cortical hyaline droplets in Groups 2 (5/5 males) and 3 (4/5 males). In Group 1 this was seen in 2/5 males at a minimal degree. In this study the occurrence of cortical hyaline droplets was accompanied by a slightly increased incidence and severity of corticomedullary tubular basophilia in Groups 2 (3/5 animals) and 3 (4/5 animals). No tubular basophilia was recorded in males of Group 1 (control).
Bone marrow-sternal: Myeloid atrophy in the sternal bone-marrow in Group 3 in 3/5 males and 3/5 females.

The staging of spermatogenesis of the animals of Groups 1 and 3 suggested normal spermatogenesis; all stages were present.

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both, control and treated rats.
Other effects:
no effects observed
Description (incidence and severity):
Upon investigation of sperm motility no abnormalities up to 250 mg/kg bw/day were seen.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
LOAEL
Effect level:
250 - 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Selected clinical chemistry parameters for females

control  100 mg/kg bw 250 (500) mg/kg bw
ALAT (U/L) 41.4 63.5 * 59.9
ASAT (U/L) 69 75.9 67.6
ALP (U/L) 67 93 78
Urea (mmol/L) 9.8 8.5 9.5

Table 2: Selected clinical chemistry parameter for males

  control  100 mg/kg bw 250 (500) mg/kg bw
ALAT (U/L) 43.2 47.7 53.4
ASAT (U/L) 77 71.3 83.4
ALP (U/L) 173 154 150
Urea (mmol/L) 10.6 8.6 7.2*

Table 3a and b: Absolute body and liver weights for males and females

 males control  100 mg/kg bw 250 (500) mg/kg bw
body weights (g) 338 331 320
liver weights (g) 8.63 9.78 ** 10.76 **

females control  100 mg/kg bw 250 (500) mg/kg bw
body weights (g) 190 197 197
liver weights (g) 5.23 6.28 * 7.63 **

Table 4a and b: Selected haematology parameters for males and females

males control  100 mg/kg bw 250 (500) mg/kg bw
Haemoglobin, mmol/L 9.8 9.6 9.4
APTT, s 16.9 18.2 16.2

Females control  100 mg/kg bw 250 (500) mg/kg bw
Haemoglobin, mmol/L 9.5 9.2 9.0*
APTT, s 16.9 18.7 13.9*

Table 5: Sperm Motility Summary

control 100 mg/kg bw 500 mg/kg bw
Motility
Mean 55 61 45
St. Dev. 22 23 26
N 5 5 5
Progressive Motility
Mean 32 29 24
St. Dev. 12 10 16
N 5 5 5

Applicant's summary and conclusion

Conclusions:
Clear toxicological effects were observed in the 500 mg/kg bw/day dose group at 9 days, this group was subsequently placed on a 5 day recovery period and resumed dosing at 250 mg/kg bw/day for the remainer of the test duration. None adverse effects (haematology, clinical biochemistry, gross necropsy, and organ weight) were observed in the 100 mg/kg bw/day group. The 28 day repeat dose NOAEL for systemic toxicity was therefore set at 100 mg/kg bw/day.
Executive summary:

In this study conducted to generally accepted scientific standards with GLP, the test material (EC 404-360-3) was administered daily via gavage (using propylene glycol as a vehicle) to rats of both sexes in doses of 0, 100 amd 500 mg/kg bw/day. Due to severe toxicity after 9 days, the 500 mg/kg bw/day was stopped for 5 days, before the same animals were re-dosed with a lower amount of 250 mg/kg bw/day for 28 days. Toxic effects were still observed at this level but not at the 100 mg/kg bw/day dose. Thus, the test material was determined to have a NOAEL of 100 mg/kg bw/day and a LOAEL of 250-500 mg/kg bw/day, however, it is not possible to completely exclude that the toxic effects observed at the 250 mg/kg bw/day was due to the previous dosing of 500 mg/kg bw/day.