Registration Dossier

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:
30 December 2014 - 18 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted to GLP with no significant deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

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 (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Montelukast Backbone Diol
- Substance type: Pharmaceutical intermediate
- Physical state: solid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): N/A
- Composition of test material, percentage of components: N/A
- Isomers composition: N/A
- Purity test date:
- Lot/batch No.: 236-4
- Expiration date of the lot/batch: 29-05-2016

- Stability under test conditions: Formulations at the entire range were stable when stored at room temperature protected from light for at least 6 hours.
- Storage condition of test material: At room temperature protected from light.
- Other:

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: Approximately 11 (males) -12 (females) weeks.
- Weight at study initiation:
- Fasting period before study:
- Housing: Pre-mating Animals were housed in groups of 5 animals of the same sex in Macrolon plastic cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum):Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: To: 23/01/2015- 18/03/2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% aqueous
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
- Concentration in vehicle: 6mg/ml, 25 mg/ml and 100mg/ml
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (27 January 2015), according to a validated method (Project 507903). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature protected from light was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. 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%.

Duration of treatment / exposure:
Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-54 days, i.e. during 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). Female nos. 41 (Group 1), 62, 63 (Group 3), 71, 72, 75, 78 and 80 (Group 4) were not dosed on the last day of their post-coitum period (post-coitum Day 21, 22 or 23) because these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/ day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
125 mg/kg bw /day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 (five)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon the results of the dose range finding study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applcable
- Section schedule rationale (if not random): Not applicable- all females allowed to litter normally.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in 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. These clinical observations were made after dosing at no specific time point, but within a similar time period after dosing for the respective animals, as there was no peak occurrence of clinical signs after dosing in the dose range finding study. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

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 :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: 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
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment period
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters checked in table [1] were examined.
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.

The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters checked in table [2] were examined.


URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

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 from lactation Day 4 onwards (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) at no specific time point, but within a similar time period after dosing for the respective animals.
- Dose groups that were examined: all groups
- Battery of functions tested: s-hearing ability, pupillary reflex and static righting reflex / -fore- and hind-limb grip strength/ motor activity

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3): All males and the selected females (5 or 6 per group, see allocation) were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.

All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Necropsy was conducted on the following days:

Females which delivered: Lactation Days 5-7.
Females which failed to deliver1 (nos. 43, 50, 61, 65, 66, 70): Post-coitum Days 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Female with total litter loss (no. 41): Within 24 hours of litter loss.
Males (including no. 75 which died after blood sampling prior to planned necropsy): Following completion of the mating period (a minimum of 28 days of dose administration).

The numbers of former implantation sites and corpora lutea were recorded for all paired females.


HISTOPATHOLOGY: Yes (see table 3)
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Histopathology
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 or 6 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 that failed to sire to examine staging of spermatogenesis and histopathology of interstitial cell structure.
- All gross lesions of all animals (all dose groups).
- The thyroid gland, jejunum, liver and kidneys of all selected males and females of Groups 2 and 3, and the thymus and spleen of all selected females of Groups 2 and 3 based on (possible) treatment-related microscopic findings in these organs.
- The mammary gland for female no 41 that had a total litter loss.
- The reproductive organs* of all males that failed to sire and all females that failed to deliver healthy pups:(Table 4). * Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings. A peer review on the histopathology data was performed by a second pathologist.





Other examinations:
Organ weights:
Terminal body weights were recorded from all males and the selected 5 females/sex/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group:
Adrenal glands Spleen
Brain Testes
Epididymides Thymus
Heart Uterus (including cervix)
Kidneys Prostate1
Liver Seminal vesicles including coagulating glands1
Ovaries Thyroid including parathyroid1
1 Weighed when fixed for at least 24 hours.

All remaining males:
Epididymides
Testes

6.8.2. Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; 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 (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
piloerectionall females at 500 mg/kg and in four females at 125 mg/kg
Mortality:
mortality observed, treatment-related
Description (incidence):
piloerectionall females at 500 mg/kg and in four females at 125 mg/kg
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight (500mg/kgbw/day females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced food consumption (500mg/kgbw/day females
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Females at 500 mg/kg had statistically significantly higher plasma levels of ALAT, ALP, albumin, cholesterol and potassium
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):
Liver weight (absolute and relatively to body weight) was dose-dependently and statistically significantly increased in males at 125 and 500 mg/kg
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The liver was enlarged in 1/10 males (microscopic correlate: hepatocellular hypertrophy) and dark red discoloration of the liver was noted in 2/10 females
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were present in the thyroid gland, jejunum, liver and kidneys of both sexes and in the thymus and spleen of females.
Histopathological findings: neoplastic:
no effects observed
Details on results:
7. RESULTS
7.1. Analysis of dose preparations
The concentrations analyzed 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%). No test substance was detected in the Group 1 formulation.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature protected from light for at least 6 hours.

7.2. Observations parental animals
For further detail on summary data, see APPENDIX 1 and on individual data, see APPENDIX 2.

7.2.1. Mortality
One female at 500 mg/kg (no.75) died on the scheduled day of necropsy shortly after blood sampling. Histopathological examination of this female did not reveal findings which could explain the death of this animal. Therefore, this death was considered to be related to the anaesthesia/blood sampling procedure and not directly related to the treatment with the test substance.

One female of the control group (no. 41) was terminated early (on Day 1 of lactation) because of total litter loss.

7.2.2. Clinical signs
Male rats showed no abnormalities.

Piloerection occurred in all females at 500 mg/kg and in four females at 125 mg/kg. This treatment-related clinical sign was first seen after about two (500 mg/kg) or three (125 mg/kg) weeks of treatment.

The only other findings noted incidentally were alopecia in one control female and scabs in the neck in one female at 125 mg/kg. These background findings were not related to treatment.

7.2.3. Functional observations
Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment.

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. It was noted that the mean values for total movements and ambulations were lower at 500 mg/kg. The differences from controls were not statistically significant and the animals at 500 mg/kg showed normal activity profiles. In males the differences were particularly due to high values in one male of the control group and low values in one male at 500 mg/kg. Therefore, these findings were considered not to be toxicologically relevant.

7.2.4. Body weights
Male rats showed no treatment-related changes in body weight or body weight gain.

Females at 500 mg/kg had statistically significantly lower body weights than controls from Day 1 of the mating period until the end of the study. Body weight gain of females at 500 mg/kg was statistically significantly lower at Day 8 of the premating period and Day 1 of the mating period. During gestation, females at 500 mg/kg had lower body weight gain from post-coitum Day 11 onwards (the differences from controls were not statistically significant). Between Days 1-4 of lactation, weight gain at 500 mg/kg was not adversely affected.

Females at 125 mg/kg had statistically significantly lower body weight gain at Day 8 of the premating period and Day 1 of the mating period. Mean body weights of these females were slightly lower compared to controls during gestation but the differences were not statistically significant except on post-coitum Day 4.

7.2.5. Food consumption
Male rats showed no treatment-related changes in food consumption before or after allowance for body weight.

In females at 500 mg/kg food consumption before or after allowance for body weight was lower during the premating period and during gestation (differences were statistically significant on most occasions during gestation). Between Days 1-4 of lactation, food consumption before allowance for body weight was similar to that in controls, whereas food consumption after allowance for body weight was somewhat higher (not statistically significantly) due to the lower body weight of females at 500 mg/kg.

Food consumption of females at 125 mg/kg was slightly lower compared to controls during the premating period (before and after allowance for body weight). Food consumption before allowance for body weight at 125 mg/kg remained slightly lower than in controls until the end of gestation but the differences were not statistically significant.

7.2.6. Haematology
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Males at 500 mg/kg had statistically significantly higher values for haemoglobin and hamatocrit. The differences from the control group were slight and not accompanied by relevant changes in other red blood cell parameters. Moreover, a decrease rather than an increase in these parameters is expected in case of a toxic effect on red blood cells. Therefore, no toxicological significance was attributed to these findings.

7.2.7. Clinical biochemistry
Females at 500 mg/kg had statistically significantly higher plasma levels of ALAT, ALP, albumin, cholesterol and potassium. Total protein in females at 500 mg/kg was also higher but the difference from controls was not statistically significant.

Clinical biochemistry values in males at 500 mg/kg did not differ statistically significantly from control values. Males at 30 and/or 125 mg/kg had a few values which were statistically significantly higher than in controls, namely: total protein and bile acids at 125 mg/kg, and cholesterol at 30 and 125 mg/kg. Cholesterol was also higher at 500 mg/kg but not statistically significantly. Mean cholesterol values in treated males were at the upper end of the historical control range1. The differences in total protein and bile acids were not attributed to treatment because there was no dose-related response. The cholesterol values showed no clear dose-related response either, but the higher values in males given the test substance could be related to treatment considering the treatment-related increase in cholesterol in females and the effect on liver weight and morphology in both sexes.

1 Historical control data for cholesterol in male rats of the strain and age used in this study:
mean of 395 male rats = 1.80 mmol/L; P5 = 1.33 mmol/L; P95 = 2.36 mmol/L.

7.2.8. Macroscopic examination
There were a few treatment-related macroscopic findings at 500 mg/kg/day. The liver was enlarged in 1/10 males (microscopic correlate: hepatocellular hypertrophy) and dark red discoloration of the liver was noted in 2/10 females (no microscopic correlate). In addition, the thymus of one female was reduced in size (microscopic correlate: lymphoid depletion) and one female at 500 mg/kg/day was emaciated.

Other incidental findings among control and treated animals were 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. These necropsy findings were therefore considered to be unrelated to treatment.

7.2.9. Organ weights
Mean terminal body weight of females at 500 mg/kg was statistically significantly lower than that of controls (relative difference: 14%).

There were treatment-related increases in the weights of the liver and kidneys.

Liver weight (absolute and relatively to body weight) was dose-dependently and statistically significantly increased in males at 125 and 500 mg/kg (relative differences from control for liver to body weight ratio: 14% and 25%, respectively). Liver to body weight ratio in females was increased, statistically significantly, by 32% at 500 mg/kg. Absolute liver weight in females at 500 mg/kg was also increased (by 13%) but not statistically significantly.

Kidney weight (absolute and relative to body weight) was statistically significantly increased in males at 500 mg/kg (relative difference from controls: 16%).

Other organ weight values in treated animals achieving a level of statistical significance when compared to controls were noted in females and consisted of a lower absolute spleen weight at 500 mg/kg and higher values for relative brain weight at 500 mg/kg, absolute thyroid weight at 30 mg/kg, relative thyroid weight at 30 and 500 mg/kg, relative adrenal weight at 500 mg/kg, and relative uterus weight at 30 mg/kg. These differences were either due to the lower terminal body weight in females at 500 mg/kg (brain, adrenals, spleen) or unrelated to treatment because there was no dose-related response (thyroid, uterus).

The other organ weights and organ to body weight ratios of treated animals were similar to those of control animals.

7.2.10. Microscopic examination
Test item-related microscopic findings were present in the thyroid gland, jejunum, liver and kidneys of both sexes and in the thymus and spleen of females.

Thyroid gland:
A slightly increased incidence and severity of follicular cell hypertrophy was recorded in males at 125 mg/kg and in both sexes at 500 mg/kg:
At 125 mg/kg this was seen in 4/5 males (3 minimal, 1 slight) and at 500 mg/kg in 4/5 males (3 minimal, 1 slight) and in 3/6 females (2 minimal, 1 slight). In one male at 500 mg/kg this was accompanied by minimal alteration of the colloid contents of the follicles. The microscopic findings for the thyroid gland are summarized in text table 1 below.

Text Table 1. Summary Test Item-Related Microscopic Thyroid gland Findings - see below


Jejunum:
In the jejunum low grades of lymphangiectasis were recorded:
At 125 mg/kg this was recorded in 1/5 males at a minimal degree and at 500 mg/kg this was seen in 4/5 males (2 minimal, 2 slight) and in 4/6 females (3 minimal, 1 slight). The microscopic finding for the jejunum is summarized in text table 2 below.

Text Table 2. Summary Test Item-Related Microscopic Jejunum Findings- see below

Liver:
A dose related increased incidence and severity of centrilobular hepatocellular hypertrophy was recorded in males starting at 30 mg/kg and in females starting at 125 mg/kg:
At 30 mg/kg this was recorded at a minimal degree in 4/5 males, at 125 mg/kg this was seen in 5/5 males (3 minimal, 2 slight) and in 4/5 females (minimal) and at 500 mg/kg in 5/5 males (3 minimal, 2 slight) and in 6/6 females (1 minimal, 4 slight, 1 moderate). This was the microscopic correlate for the increased liver weights. The microscopic finding for the liver is summarized in text table 3 below.

Text Table 3. Summary Test Item-Related Microscopic Liver Findings - see below

Kidneys:
Hyaline droplet accumulation was present at a slightly increased incidence and severity in males treated at 125 and 500 mg/kg:
This was recorded at 125 mg/kg in 5/5 males (2 minimal, 3 slight) and at 500 mg/kg in 5/5 males (4 minimal, 1 slight). A background level of this finding was recorded for 2/5 males of the control Group 1 (minimal) and in 3/5 males at 30 mg/kg/day (2 minimal, 1 slight). Hyaline droplet accumulation was considered to be the microscopic correlate for the slightly increased kidney weights recorded for the treated male rats.

Tubular basophilia was recorded at a slightly increased severity (bilateral, slight) in 1/6 females at 500 mg/kg. In this animal this was accompanied by minimal tubular degeneration. A minimal degree of tubular basophilia is a common background finding and was recorded in 3/5 females of the control Group 1 and 1/5 females at 125 mg/kg.

Mineralization (tubular or papillary) was recorded in 2/5 females (minimal) at 30 mg/kg, 2/5 females (minimal) at 125 mg/kg and in 3/6 females (2 minimal, 1 slight) at 500 mg/kg.

The microscopic findings for the kidneys are summarized in text table 4 below.

Text Table 4. Summary Test Item-Related Microscopic Kidney Findings - see below


Thymus (females):
Lymphoid depletion was recorded slightly above background incidence and severity in females at 500 mg/kg:
This was recorded in 2/6 females at a slight-moderate degree at 500 mg/kg, compared to 1/5 females at a minimal degree of the control Group 1. The microscopic findings for the thymus of females are summarized in text table 5 below.

Spleen (females):
A minimal decreased severity in extramedullary hematopoiesis was present in females treated at 125 and 500 mg/kg:
This was recorded at 500 mg/kg in 6/6 females (4 minimal, 1 slight, 1 moderate, mean severity 1.5), at 125 mg/kg in 5/5 females (2 minimal, 3 slight, mean severity 1.6) compared to 5/5 in the control Group 1 (slight, mean severity 2.0) and at 30 mg/kg in 5/5 females (1 minimal, 2 slight, 2 moderate, mean severity 2.2). The microscopic findings for the spleen of females are summarized in text table 5 below.

Text Table 5. Summary Test Item-Related Microscopic thymus and spleen Findings (females)- see below

There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

There were nine couples which failed to deliver healthy pups: four in the control group, one at 30 mg/kg and four at 125 mg/kg. No abnormalities were seen in the reproductive organs, which could account for their infertility or total litter loss.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test substance and spermatogenic staging profiles were normal for all males examined.


Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Text Table 1. Summary Test Item-Related Microscopic Thyroid gland Findings

 

Males

Females

Dose level (mg/kg):

0

30

125

500

0

30

125

500

 

 

 

 

 

 

 

 

 

Thyroid glanda

5

5

5

5

5

5

5

6

  Hypertrophy, follicular cell

 

 

 

 

 

 

 

 

      Minimal

2

2

3

3

1

1

1

2

      Slight

   Alteration colloid

      Minimal

-

 

-

-

 

-

1

 

-

1

 

1

-

 

-

-

 

-

-

 

-

1

 

-

a = Number of tissues examined from each group.


 

Text Table 2. Summary Test Item-Related Microscopic Jejunum Findings

 

Males

Females

Dose level (mg/kg):

0

30

125

500

0

30

125

500

 

 

 

 

 

 

 

 

 

Jejunuma

5

5

5

5

5

5

5

6

  Lymphangiectasis

 

 

 

 

 

 

 

 

      Minimal

-

-

1

2

-

-

-

3

      Slight

-

-

-

2

-

-

-

1

a = Number of tissues examined from each group.

 

Text Table 3. Summary Test Item-Related Microscopic Liver Findings

 

Males

Females

Dose level (mg/kg):

0

30

125

500

0

30

125

500

 

 

 

 

 

 

 

 

 

Livera

5

5

5

5

5

5

5

6

  Hypertrophy hepatocellular,

  centrilobular

 

 

 

 

 

 

 

 

      Minimal

-

4

3

3

-

-

4

1

      Slight

      Moderate

-

-

-

-

2

-

2

-

-

-

-

-

-

-

4

1

a = Number of tissues examined from each group.

 

Text Table 4. Summary Test Item-Related Microscopic Kidney Findings

 

Males

Females

Dose level (mg/kg/day):

0

30

125

500

0

30

125

500

 

 

 

 

 

 

 

 

 

Kidneya

5

5

5

5

5

5

5

6

  Hyaline droplet accumulation

 

 

 

 

 

 

 

 

      Minimal

2

2

2

4

-

-

-

-

      Slight

   Basophilia, tubular

      Minimal

      Slight

   Mineralization

      Minimal

      Slight

   Degeneration tubular

      Minimal

-

 

-

-

 

-

-

 

-

1

 

2

-

 

-

-

 

-

3

 

2

-

 

-

-

 

-

1

 

-

-

 

-

-

 

-

-

 

3

-

 

-

-

 

-

-

 

-

-

 

2

-

 

-

-

 

1

-

 

2

-

 

-

-

 

-

1

 

2

1

 

1

a = Number of tissues examined from each group.

Text Table 5. Summary Test Item-Related Microscopic thymus and spleen Findings (females)

 

Females

Dose level (mg/kg/day):

0

30

125

500

 

 

 

 

 

Thymusa

5

5

5

6

  Depletion lymphoid

 

 

 

 

      Minimal

1

-

-

-

      Slight

-

-

-

1

      Moderate

-

-

-

1

 

Spleena

 

5

 

5

 

5

 

6

  Hematopoiesis extramedullary

 

 

 

 

      Minimal

-

1

2

4

      Slight

5

2

3

1

       Moderate

-

2

-

1

a = Number of tissues examined from each group.

 

Applicant's summary and conclusion

Conclusions:
Montelukast Backbone Diol was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 125 and 500 mg/kg. Concurrent controls (10 rats/sex) received the vehicle, 1% aqueous carboxymethyl cellulose, alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-54 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature protected from light.

Parental results:
Piloerection was noted in all females at 500 mg/kg and in a few females at 125 mg/kg.

There was a treatment-related decrease in body weight (gain) in females at 500 mg/kg and, to a lesser extent in females at 125 mg/kg. Body weight gain was not affected during the lactation period. Food consumption showed a similar pattern. The effect on body weight at 500 mg/kg was considered to be toxicologically relevant because mean body weights were decreased by about 10%. The slight effect on body weight at 125 mg/kg was considered not to be toxicologically significant.

Liver effects were noted at 125 and 500 mg/kg in both sexes and at 30 mg/kg in males. This was indicated by increased liver weights (at 125 and 500 mg/kg in males, at 500 mg/kg in females), centrilobular hepatocellular hypertrophy (at all dose levels in males, at 125 and 500 mg/kg in females), and changes in clinical biochemistry values (higher plasma levels of ALAT, ALP, albumin and cholesterol in females at 500 mg/kg). Furthermore, a few animals treated at 500 mg/kg showed macroscopic changes of the liver (enlarged or dark red discolored). Increased liver weight and hepatocellular hypertrophy in absence of degenerative changes is regarded as an adaptive, non-adverse response (Ref. 6) and the magnitude of the changes in clinical biochemistry values was modest. Therefore, these liver findings were considered not to be toxicologically relevant.

One female treated at 500 mg/kg had a degenerative change in the kidneys, namely slight renal tubular basophilia (bilateral) in combination with minimal tubular degeneration. This was considered to be an adverse, treatment-related change. The other treatment-related microscopic change noted in the kidneys of females consisted of tubular or papillary mineralizaton. This finding was considered not to be adverse at the incidence (2 females in each test group) and severity (mostly minimal) observed.

Treatment-related renal changes were also noted in males. At 125 and 500 mg/kg the incidence and/or severity of hyaline droplet accumulation were higher than in controls. This finding likely represented alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation. The slightly increased hyaline droplet accumulation was not accompanied by any degenerative change and was therefore considered not to be an adverse finding. This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref.7). The increase in kidney weight (absolute and relative to body weight) in males at 500 mg/kg was likely to be related to this microscopic change.

Hypertrophy of the follicular epithelium of the thyroid was observed at a slightly increased incidence and severity at 500 mg/kg in both sexes and at 125 mg/kg in males. This change is known to occur as a secondary effect associated with centrilobular hepatocellular hypertrophy and subsquent increased thyroid hormone elimination (Ref. 8). It is regarded as an adaptive, non-adverse change (Ref. 9).

Further treatment-related microscopic changes were seen in the jejunum (low grades of dilated lymphatic vessels at 125 and 500 mg/kg), thymus (lymphoid depletion at 500 mg/kg) and spleen (decreased severity of extramedullary haematopoiesis at 500 mg/kg). These changes were considered not to represent adverse effects of the test substance on these organs.

No treatment-related or toxicologically relevant changes were noted in the remaining parental parameters investigated in this study (i.e. functional observations and haematology parameters.

Reproductive results:
No reproductive toxicity was observed up to the highest dose level tested (500 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results:
At 500 mg/kg mean pups weights on Day 4 of lactation (male and female pups) were slightly (about 10%) lower. Although the differences from control values were not statistically significant, this decrease in pup body weight gain was considered to be related to treatment and of toxicological relevance. No developmental toxicity was observed at the lower dose levels (30 and 125 mg/kg).

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and the early postnatal pup development endpoints mortality, clinical signs and macroscopy).

The number of females with living pups in the control group (6/10) was lower than normal. There was no explanation for this finding. The offspring in the control group was healthy and litter size was normal. Litter size at the highest dose level tested (500 mg/kg) was also normal. It was therefore concluded that this study was adequate for a meaningful evaluation of possible developmental (from implantation onwards) toxicity of the test substance. To further support this conclusion historical control data from a large number similar studies are presented in Appendix 7 of this report.


In conclusion, treatment with Montelukast Backbone Diol by oral gavage in male and female Wistar Han rats at dose levels of 30, 125 and 500 mg/kg revealed parental toxicity at 125 and 500 mg/kg in females. Toxicologically relevant findings at 500 mg/kg included piloerection in all females, reduced body weight and food consumption, and a degenerative change in the kidneys in one female. At 125 mg/kg piloerection was noted in 4/10 females. Based on the piloerection at 125 mg/kg, the parental No Observed Adverse Effect Level (NOAEL) was placed at 30 mg/kg.

No reproductive effects or developmental effects on offspring were seen at doses below those causing maternal effects. As no reproduction toxicity was observed for treatment up to 500 mg/kg, the reproduction NOAEL was placed at at least 500 mg/kg. Based on the lower body weight gain of pups at 500 mg/kg, the developmental NOAEL was placed at 125 mg/kg.

Executive summary:

Guidelines

The study was based on the following guidelines:

1)    OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996.

2)    OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

3)    OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

4)    OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

5)    EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

6)    OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

7)    OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

 

Rationale for dose levels

Dose levels were based on a 10-day dose range finding study (Project 507905; seeAPPENDIX5) in which dose levels of 500 and 1000 mg/kg were tested. The main finding was an increase in liver weight by about one third at both dose levels.  

 

Study outline

The test substance, formulated in1% Aqueous carboxymethyl cellulose, was administered daily by oral gavage to SPF-bred Wistar Han rats at dose levels of 30, 125 and 500 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle only.Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for41-54[doc1] days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment),  body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

 

Results/discussion

 

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

 

Parental results:

Piloerection was noted in all females at 500 mg/kg and in a few females at 125 mg/kg.

 

There was a treatment-related decrease in body weight (gain) in females at 500 mg/kg and, to a lesser extent in females at 125 mg/kg. Body weight gain was not affected during the lactation period. Food consumption showed a similar pattern. The effect on body weight at 500 mg/kg was considered to be toxicologically relevant because mean body weights were decreased by about 10%. The slight effect at 125 mg/kg was considered not to be toxicologically significant.

 

Liver effects were noted at 125 and 500 mg/kg in both sexes and at 30 mg/kg in males. This was indicated by increased liver weights (at 125 and 500 mg/kg in males, at 500 mg/kg in females), centrilobular hepatocellular hypertrophy (at all dose levels in males, at 125 and 500 mg/kg in females), and changes in clinical biochemistry values (higher plasma levels of ALAT, ALP, albumin and cholesterol in females at 500 mg/kg). Furthermore, a few animals treated at 500 mg/kg showed macroscopic changes of the liver (enlarged or dark red discolored). Increased liver weight and hepatocellular hypertrophy in absence of degenerative changes is regarded as an adaptive, non-adverse response (Ref. 6) and the magnitude of the changes in clinical biochemistry values was modest. Therefore, these liver findings were considered not to be toxicologically relevant.

 

One female treated at 500 mg/kg had a degenerative change in the kidneys, namely slight renal tubular basophilia (bilateral) in combination with minimal tubular degeneration. This was considered to be an adverse, treatment-related change.

 

In the kidneys of males treated at 125 and 500 mg/kg the incidence and/or severity of hyaline droplet accumulation were increased. This finding likely representedalpha2uglobulin accumulation, a male-rat specific phenomenon, which is not relevant for humans.The increase in kidney weight (absolute and relative to body weight) in males at 500 mg/kg was likely to be related to this finding.

 

Hypertrophy of the follicular epithelium of the thyroid was observed at a slightly increased incidence and severity at 500 mg/kg in both sexes and at 125 mg/kg in males. This change is known to occur as a secondary effect associated with centrilobular hepatocellular hypertrophy and subsquent increased thyroid hormone elimination. It is regarded as an adaptive, non-adverse change.

 

The other treatment-related microscopic changes observed were considered not to represent adverse effects of the test substance on these organs (jejunum: low grades ofdilated lymphatic vessels at 125 mg/kg (males) and 500 mg/kg (both sexes);thymus of females: lymphoid depletion at 500 mg/kg; spleen of females: decreased severity of extramedullary haematopoiesis at 500 mg/kg; kidneys of females:tubular or papillary mineralizaton all dose levels).

 

No treatment-related or toxicologically relevant changes were noted in the remaining parental parameters investigated in this study (i.e. functional observations and haematology parameters.

 

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (500 mg/kg).

 

At 500 mg/kg mean pups weightson Day 4 of lactation (male and female pups) were slightly (about 10%) lower compared to control values.No developmental toxicity was observed at the lower dose levels (30 and 125 mg/kg).

 

 

Conclusion

 

In conclusion, treatment withMontelukast Backbone Diolby oral gavage in male and female Wistar Han rats at dose levels of 30, 125 and 500 mg/kg revealed parental toxicity at 125 and 500 mg/kg in females. Toxicologically relevant findings at 500 mg/kg included piloerection in all females, reduced body weight and food consumption, and a degenerative change in the kidneys in one female. At 125 mg/kg piloerection was noted in 4/10 females. Based on the piloerection at 125 mg/kg, the parental No Observed Adverse Effect Level (NOAEL) was placed at 30 mg/kg.

 

No reproductive effects or developmental effects on offspring were seen at doses below those causing maternal effects. As no reproduction toxicity was observed for treatment up to 500 mg/kg, the reproduction NOAEL was placed at at least 500 mg/kg. Based on the lower body weight gain of pups at 500 mg/kg, the developmental NOAEL was placed at 125 mg/kg.