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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
17 Feb - 07 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: light yellow transparent oily liquid
- Batch: H113021
- Analytical purity: 100%
- Storage conditions: at room temperature in the dark
- Stability under storage: stable
- Expiry date: November 2010
- Density: 0.944

Test animals

Species:
rat
Strain:
other: Crl:WI (Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277 - 353 g (males) and 183 - 225 g (females)
- Housing: 5 animals of the same sex per cage in Macrolon cages (MIV type). Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenised to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

DOSE VOLUME:
5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 h.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 5 females (main study)
5 males and 5 females (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 10-day dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males: Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.
Sacrifice and pathology:
DAY OF NERCROPSY:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanised in extremis.

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
From the first 5 Main animals/sex/group, all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):
Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

From all remaining animals and females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.











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 if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test 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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg bw/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg bw/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg bw/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg bw/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time in males at 1000 mg/kg bw/day and lower relative lymphocyte counts in females at 300 mg/kg bw/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg bw/day, and higher chloride levels in males at 100 mg/kg bw/day. Changes at 1000 mg/kg bw/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg bw/day had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg bw/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg bw/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg bw/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg bw/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg bw/day and the end of treatment, lower heart weight in females at 1000 mg/kg bw/day, higher heart to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg bw/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg bw/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discolouration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discolouration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg bw/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Effect levels

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: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion