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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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:
23 July 2012 to 2 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Appearance: dark brown, viscous liquid
- Storage conditions: room temperature in the dark
- Stability under storage conditions: stable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality).
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: The average weight of the males was 326 g and the average weight of the females was 206 g on Day 1 of the study.
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis in plastic cages (height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During lactation, pups were kept with the dam until termination in plastic cages (height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle

IN-LIFE DATES: From: 9 August 2012 To: 2 October 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Specific gravity: 1.036
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No correction was made for the purity of the test material. Adjustment was made for specific gravity of the vehicle. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the test material in formulations.
Samples of dose preparations were taken on a single occasion during the treatment phase and were stored and dispatched on dry ice to the test site for formulation analysis.
Formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 5 hours was also determined (highest and lowest concentration).

Specific sampling instructions:
− Appearance of the formulations was recorded prior to sampling
− Formulations were placed on a magnetic stirrer during sampling
− Sampling was conducted with a clean pipette tip used for every group
− Duplicate samples were taken from each sampling height, with a sample volume of 0.5 mL
− Samples were weighed (grams) on an analytical balance at a precision of 4 decimals
− T, M and B samples were stored on dry ice immediately after sampling
− S (Stability) samples were kept at room temperature under normal laboratory light conditions for 5 hours, and then placed on dry ice
− All samples were transferred to the weighing room at once for dispatch

SAMPLES
In total, 20 samples were included, randomised over 4 formulation groups. Group 1 was the control group. Group 2, 3 and 4 were dosed at 75, 250 and 750 mg/kg bw/day, respectively. The samples of the control group and group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice.
Samples of treatment groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container. In addition, stability samples were taken from formulation groups 2 and 4, in duplicate at the middle position of the container, stored for 5 hours at room temperature under normal laboratory light conditions and then placed on dry ice.
The samples were stored at the analytical facility at a target temperature ≤-70 °C.

ANALYTICAL METHOD
The determination was performed using LCMS/MS.

SAMPLE INJECTIONS
The analytical run was acquired in the following order:
- Analytical blank sample
- Calibration curve
- Analytical blank sample
- Analytical blank sample
- Procedural recovery sample low and high (replicate 1)
- Analytical samples
- Stability at storage conditions low and high (replicate 1 and 2)
- Procedural recovery samples low and high (replicate 2)
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

ELECTRONIC DATA CAPTURE
System control, data acquisition and data processing were performed using the program Analyst version 1.5.2 (Applied Biosystems, Burlington, Canada).
Electronic raw data was stored in the document management system using KnowledgeTree version 3.5.2c (Raleigh, USA).

FORMULAS
Response (Y)
Peak area test substance [cps]

Calibration curve
Y = bX + a
where:
X = nominal concentration [mg/L]
b = slope [cps × L/mg]
a = intercept [cps]

Analysed concentration (X)
X = ((Y - a) / b) x ((V x d) / w [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
(Y - a) / b values are obtained from Analyst

Recovery
(Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy
(Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.)
((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at the retention time of the test substance should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).

Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration and if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the absolute relative difference between the stored and freshly taken samples was ≤ 10 %.

RESULTS
Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Linear regression analysis was performed using the least squares linear regression with a 1/concentration² weighting factor.
The coefficient of correlation (r) of the calibration curve was 0.9998.
Additionally, there was no response in all analytical blank samples at the retention time of the test material.

Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.

Test samples
In the Group 1 formulation, no test material was detected. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 and Group 4 formulations after storage yielded an absolute relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Stability at storage conditions
It was observed that the mean accuracy was 94.9 and 98.2 % at the levels high and low, respectively, which is within the criteria set (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of 29 days at a temperature of ≤-70 °C.
Duration of treatment / exposure:
Males were exposed for 28 days: 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 55 days: 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 250 and 750 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study conducted at the testing laboratory.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality / viability at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing (at no specific time point, but around the same time point for all animals). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated 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
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes. The animals were deprived of food overnight (maximum of 24 hours) before blood sampling, but water was provided.
- How many animals: Blood samples were collected from 5 randomly selected animals/sex/group. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with EDTA (0.5 mL).
- Parameters examined: 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 and platelets.
- Instrumentation: ADVIA 120

CLINICAL CHEMISTRY: Yes
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes. The animals were deprived of food overnight (maximum of 24 hours) before blood sampling, but water was provided.
- How many animals: Blood samples were collected from 5 randomly selected animals/sex/group. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with Li-heparin (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate and bile acids.
- Instrumentation: Olympus AU400

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period. These tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals and before blood sampling.
- Dose groups that were examined: The tests were performed on 5 randomly selected animals/sex/group.
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and locomotor activity (total movements and ambulations).

OTHER: CLOTTING POTENTIAL
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes. The animals were deprived of food overnight (maximum of 24 hours) before blood sampling, but water was provided.
- How many animals: Blood samples were collected from 5 randomly selected animals/sex/group. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with citrate (0.45 mL).
- Parameters examined: Prothrombin time and activated partial thromboplastin time.
- Instrumentation: STA Compact
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy of parental animals: All males and 5 selected females/group for full examination, though 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. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
- Sacrifice: All males and the 5 selected females/group were deprived of food overnight (maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using isoflurane and subsequently exsanguinated.
- Necropsy of pups: Pups surviving to planned termination were killed by decapitation on Days 5 to 7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected and fixed: adrenal glands, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) 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, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.

Samples of the following tissues and organs were collected and fixed from all remaining animals and females which failed to deliver: cervix, clitoral gland, coagulation gland, epididymides, mammary gland area, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina and all gross lesions.

Organ weights: The following organ weights and terminal body weight were recorded from the 5 randomly selected animals/sex/group on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles including coagulating glands and thyroid including parathyroid.

The following organ weights and terminal body weight were recorded from all remaining males: epididymides and testes.

All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 micrometers and stained with haematoxylin and eosin. From the selected 5 males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3 to 4 micrometers, and stained with PAS/haematoxylin.

A peer review on the histopathology data was performed by a second pathologist.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the randomly selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups).
- Liver (both sexes), kidney (males) and thyroids (females) of the 5 randomly selected animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs* of all males that failed to sire and all females that failed to deliver healthy pups. Additionally, the reproductive organs from two males were examined since the female with whom they were paired were killed in extremis or died spontaneously.

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
Other examinations:
Further parameters examined in the parental generation as part of the screening for reproductive and developmental effects were: mating date, confirmation of pregnancy and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined. Reproductive indices were also calculated.
Observations in the F1 generation included: the numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated. At least once daily, detailed clinical observations were conducted for all animals. Live pups were weighed on Days 1 and 4 of lactation. Sex was determined for all pups on Days 1 and 4 of lactation. Offspring viability indices were calculated.
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.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup 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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
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):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
One female at 250 mg/kg was euthanised in extremis after 26 days of treatment; no definitive cause of death could be determined.
One female at 750 mg/kg was found dead after 33 days of treatment. Macroscopic examination confirmed that her death was secondary to a gavage error, and was in no way related to treatment.

CLINICAL SIGNS
Clinical signs noted for the female at 250 mg/kg that was euthanised in extremis included lethargy, hunched posture, laboured or shallow respiration, rales, piloerection, lean appearance and salivation. These were noted for several days prior to her death.
There were no other toxicology relevant observations noted up to 750 mg/kg.
Salivation was noted for all males and females at 250 and 750 mg/kg. Salivation was also noted for all males and 3/10 females at 75 mg/kg. Salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to the taste of the test material.
Pale faeces were transiently noted for 4 females at 750 mg/kg. No toxicological relevance was attributed to this finding as it was transient and was not accompanied by any other findings.
Incidental findings that were noted included scabs and swelling of the right ear noted for control animals.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

BODYWEIGHTS
Males at 750 mg/kg had significantly lower body weight gains on Days 8 and 14 of the mating period. As the difference from controls was only slight, this was not considered to be adverse.
All other body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
There were no toxicologically relevant effects on absolute or relative food consumption noted up to 750 mg/kg.
There was statistically significant increase in absolute food consumption for females at 750 mg/kg over post coitum Days 4 to 7. Absolute and relative food consumption was significantly increased for females at 75 mg/kg over post coitum Days 14 to 17. This was not considered toxicologically relevant since the difference from controls was only slight and/or occurred in the absence of a treatment related distribution.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Females at 750 mg/kg had significantly lower neutrophils and significantly increased lymphocyte counts than controls. This was likely secondary to slightly high neutrophil and low lymphocyte means obtained for control animals, and was not considered to be toxicologically relevant.
Minor statistically significant differences arising between controls and animals receiving 250 mg/kg were considered not to represent a change of biological relevance.

CLINICAL CHEMISTRY
At 750 mg/kg the following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from controls:
-Increased alanine aminotransferase; females.
-Increased alkaline phosphatase; both sexes. Also seen for animals at 250 mg/kg, but was significant for males only.
-Reduced total bilirubin; males. Also seen at 75 and 250 mg/kg without dose response.
-Increased glucose; males.
-Lower cholesterol; males. Also seen at 75 and 250 mg/kg.
-Increased inorganic phosphate; males.

MACROSCOPIC EXAMINATION
Macroscopic findings noted for the female at 250 mg/kg that was euthanised in extremis included enlarged mandibular lymph node, thymus reduced in size and GI tract distended with gas.
Findings noted for the female at 750 mg/kg that died due to a gavage error included fluid in the pericardium, perforation of the oesophagus, both adrenals enlarged and many dark red foci on the thymus. In the uterus, 1 foetus and 4 early resorptions were seen in the right uterine horn and 7 foetuses and 1 early resorption were seen in the left uterine horn.
At 750 mg/kg all males and 7/10 females had visibly enlarged livers at the macroscopic examination.
Enlarged liver was also noted for 2 females at 250 mg/kg. This was considered toxicologically relevant.
One female at 250 mg/kg was found with 1 mummified foetus and dark red and watery clear fluid in the uterus. This female did not deliver any live pups.
Incidental findings noted for control and treated animals included enlarged mandibular lymph nodes, yellowish-soft nodule or nodules on the epididymis tail, pelvic dilation of one or both kidneys, enlarged spleen, ectopic splenic tissue, reddish or dark red discolouration of the mandibular or renal lymph nodes, tan focus on the left clitoral gland, yellowish, greenish hard nodule on epididymal adipose tissue, reduced size of the thymus, and alopecia on various body regions. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. As such, these were not considered to be toxicologically relevant.

ORGAN WEIGHTS
At 750 mg/kg terminal body weights were significantly lower for males while significantly increased absolute and relative liver weights (both sexes) were seen. Absolute and relative liver weights were significantly higher for both sexes at 250 mg/kg as well.
Kidney to body weight ratios were significantly higher for males of all treatment groups, but in the absence of any adverse renal findings noted at the microscopic examination, it was not considered to be biologically significant.
Absolute and relative thyroid weights were also significantly higher for males of all treatment groups.
However, the values obtained for controls were slightly low as weights at 75 mg/kg matched the historical mean (mean absolute thyroid weight = 0.029, mean relative thyroid weight = 0.008). As such, the significant differences were not considered to be toxicologically relevant. The lack of thyroid effects for males seen at the histopathological examination also supports this conclusion.
Absolute heart weights were significantly lower for males at 250 mg/kg compared to controls. In the absence of a treatment related distribution, it was not considered to be toxicologically relevant.

MICROSCOPIC EXAMINATION
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material.
There were treatment-related microscopic findings present in the liver, thyroid gland and kidneys.

Liver
-Centrilobular hypertrophy of the hepatocytes (above background) was present in female rats treated at 250 mg/kg (4/7 rats) up to a slight degree and in male and female rats treated at 750 mg/kg (10/10 and 9/9 rats respectively) up to marked degree.

Thyroid glands
-Hyperplasia and hypertrophy, follicular was present at increased incidence and severity in females treated at 750 mg/kg (5/6) compared to control (1/5), 75 mg/kg (1/5) and 250 mg/kg (0/5) treated rats.

Kidneys
-Hyaline droplets were present at an increased incidence and/or severity in male rats treated at 250 (Group 3; 5/5) and 750 mg/kg (Group 4; 5/5), compared to control (3/5) and 75 mg/kg (Group 2; 5/7) treated males.
The cortical hyaline droplets, representing alpha2µglobulin, were not accompanied by an increase in indicators of tubular damage and the severity grades were within the range seen in untreated male rats.

Effect levels

Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Summary of Selected Clinical Chemistry Parameters - End of Treatment

 

 

Dose Level (mg/kg)

Males

Females

Control

75

250

750

Control

75

250

750

Alanine Aminotransferase

(U/L)

Mean

SD

N

44.3

6.1

5

44.0

6.3

5

40.7

8.9

5

47.3

10.1

5

43.3

12.7

5

47.1

10.0

5

55.4

14.0

5

73.4**

10.0

5

Alkaline

Phosphatase

(U/L)

Mean

SD

N

140

12

5

168

22

5

234*

67

5

316**

58

5

85

34

5

94

16

5

111

60

5

223*

119

5

Total Bilirubin

(µmol/L)

Mean

SD

N

2.4

0.3

5

2.0**

0.2

5

1.8**

0.1

5

1.9**

0.1

5

2.0

0.3

5

1.8

0.3

5

1.7

0.2

5

1.8

0.1

5

Glucose

(mmol/L)

Mean

SD

N

7.46

0.65

5

8.76

2.27

5

9.82

0.17

5

10.61*

1.98

5

6.40

1.00

5

6.80

1.11

5

7.02

1.42

5

7.11

0.99

5

Cholesterol

(mmol/L)

Mean

SD

N

1.81

0.29

5

1.36**

0.12

5

1.24**

0.23

5

0.94**

0.14

5

1.27

0.15

5

1.73

0.44

5

1.42

0.30

5

1.60

0.39

5

Inorganic Phosphate

(mmol/L)

Mean

SD

N

2.17

0.12

5

1.93

0.22

5

2.19

0.22

5

2.05*

0.05

5

2.25

0.29

5

2.21

0.35

5

2.59

0.99

5

2.59

0.61

5

*Dunnett-test based on pooled variance significant at 5 % level

**Dunnett-test based on pooled variance significant at 1 % level

SD = Standard deviation

 

Table 2 Summary of Selected Organ Weights- End of Treatment

 

 

Dose Level (mg/kg)

Males

Females

Control

75

250

750

Control

75

250

750

Bodyweight

(g)

Mean

SD

N

361

12

10

361

18

10

349

11

10

343*

9

10

244

2

5

249

10

5

242

12

5

245

19

5

Heart

(g)

Mean

SD

N

0.998

0.080

5

0.972

0.050

5

0.883*

0.071

5

0.915

0.058

5

0.793

0.053

5

0.797

0.077

5

0.763

0.057

5

0.794

0.131

5

Liver

(g)

Mean

SD

N

8.86

0.64

5

9.73

0.71

5

11.63**

0.71

5

15.71**

1.63

5

7.44

0.43

5

8.32

0.47

5

9.11**

0.78

5

11.83**

0.69

5

Thyroids

(g)

Mean

SD

N

0.019

0.002

5

0.029**

0.004

5

0.027*

0.006

5

0.032**

0.005

5

0.023

0.004

5

0.022

0.003

5

0.025

0.005

5

0.024

0.004

4

Kidneys

(g)

Mean

SD

N

2.24

0.20

5

2.45

0.18

5

2.53

0.22

5

2.50

0.17

5

1.72

0.16

5

1.83

0.08

5

1.76

0.16

5

1.79

0.08

5

*Dunnett-test based on pooled variance significant at 5 % level

**Dunnett-test based on pooled variance significant at 1 % level

SD = Standard deviation

 

Table 3 Summary of Selected Organ/Bodyweight Ratios - End of Treatment

 

 

Dose Level (mg/kg)

Males

Females

Control

75

250

750

Control

75

250

750

Bodyweight

(g)

Mean

SD

N

361

12

10

361

18

10

349

11

10

343*

9

10

244

2

5

249

10

5

242

12

5

245

19

5

Heart

(%)

Mean

SD

N

0.276

0.014

5

0.273

0.007

5

0.254

0.023

5

0.269

0.015

5

0.325

0.024

5

0.321

0.030

5

0.315

0.018

5

0.322

0.030

5

Liver

(%)

Mean

SD

N

2.45

0.11

5

2.74

0.17

5

3.34**

0.16

5

4.61**

0.44

5

3.05

0.17

5

3.36

0.28

5

3.76**

0.32

5

4.84**

0.35

5

Thyroids

(%)

Mean

SD

N

0.005

0.001

5

0.008**

0.001

5

0.008*

0.001

5

0.009**

0.001

5

0.009

0.002

5

0.009

0.001

5

0.010

0.002

5

0.010

0.002

4

Kidneys

(%)

Mean

SD

N

0.62

0.04

5

0.69*

0.03

5

0.73**

0.04

5

0.73**

0.04

5

0.71

0.07

5

0.74

0.05

5

0.73

0.06

5

0.73

0.03

5

*Dunnett-test based on pooled variance significant at 5 % level

**Dunnett-test based on pooled variance significant at 1 % level

SD = Standard deviation

 

Table 4 Summary of Macroscopic Findings - Males

 

Dose Level (mg/kg)

Control

75

250

750

End of Treatment

Animals examined

Animals without findings

Animals affected

10

8

2

10

8

2

10

5

5

10

0

10

Liver

Enlarged

Kidneys

Pelvic dilation

Epididymides

Nodule(s)

Spleen

Ectopic splenic tissue

Enlarged

Thymus

Reduced in size

Mandibular lymph node

Enlarged

Discolouration

Renal lymph node

Discolouration

Body cavities

Nodule(s)

 

0

 

0

 

1

 

0

0

 

0

 

1

0

 

0

 

0

 

0

 

2

 

0

 

1

0

 

0

 

0

0

 

0

 

0

 

0

 

1

 

1

 

0

1

 

0

 

0

1

 

1

 

1

 

10**

 

0

 

1

 

0

0

 

1

 

0

0

 

0

 

0

**Fisher’s Exact test significant at 1 % level

 

Table 5 Summary of Macroscopic Findings - Females

 

Dose Level (mg/kg)

Control

75

250

750

Intercurrent Death

Animals examined

Animals affected

 

 

1

1

1

1

General observations

Gi−tractus: distended with gas

Heart

Contains fluid

Oesophagus

Perforation(s)

Liver

Enlarged

Uterus

Contents

Adrenal glands

Enlarged

Thymus

Focus/foci

Reduced in size

Mandibular lymph node

Enlarged

 

 

 

1

 

0

 

0

 

0

 

0

 

0

 

0

1

 

1

 

0

 

1

 

1

 

1

 

1

 

1

 

1

0

 

0

End of Treatment

Animals examined

Animals without findings

Animals affected

10

9

1

10

9

1

9

5

4

9

3

6

Liver

Enlarged

Kidneys

Pelvic dilation

Uterus

Left horn: one mummified foetus

Contains fluid

Contents

Clitoral Glands

Focus/foci

Spleen

Enlarged

Skin

Alopecia

 

0

 

0

 

0

0

0

 

0

 

0

 

1

 

0

 

0

 

0

0

0

 

1

 

0

 

0

 

2

 

1

 

1

1

1

 

0

 

0

 

1

 

6**

 

0

 

0

0

0

 

0

 

1

 

0

**Fisher’s Exact test significant at 1 % level

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL was determined to be 75 mg/kg bw/day for repeated dose toxicity. However as no significant or severe toxic effects were seen in the 250 mg/kg bw/day dose group, no classification for specific target organ toxicity is required in accordance with EU criteria.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.

Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 75, 250 and 750 mg/kg/day in propylene glycol. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 41 to 55 days (2 weeks prior to mating, during mating, during postcoitum and during at least 4 days of lactation).

Animals were evaluated for mortality/viability, clinical signs, functional observations and locomotor activity, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.

Changes in clinical biochemistry parameters, increased organ weights, macroscopically enlarged livers and microscopic findings in the liver (centrilobular hepatocytic hypertrophy) and thyroids (follicular hyperplasia and hypertrophy) characterised parental toxicity at 750 mg/kg. Higher liver weights and liver to body weight ratios were seen for both sexes and centrilobular hypertrophy of the liver was also seen for females at 250 mg/kg.

No toxicologically relevant changes were noted in any of the remaining parental parameters.

Under the conditions of this study, the NOAEL was determined to be 75 mg/kg bw/day for repeated dose toxicity. However as no significant or severe toxic effects were seen in the 250 mg/kg bw/day dose group, no classification for specific target organ toxicity is required in accordance with EU criteria.