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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-12-2014 to 18-03-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted according to GLP with no major deviations

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(1S)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propanol
EC Number:
608-251-3
Cas Number:
287930-77-2
Molecular formula:
C29H28NO2Cl
IUPAC Name:
(1S)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propanol
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
Details on test animals or test system 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 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%.

Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
In first instance detection of mating was not confirmed for animal nos. 61 and 67. Re-evaluation of lavages on Day 6 of the mating period showed evidence of mating in the lavage of the first day of mating. These females were separated from the males on Day 6 of mating.
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.
Duration of test:
54 days (female) , 31 days (male)
Doses / concentrationsopen allclose all
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

Maternal examinations:
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

GENERAL REPRODUCTION DATA: Male number paired with, 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.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other:
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
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.


Indices:
Mating index (%) Number of females mated x 100
Number of females paired

Fertility index (%) Number of pregnant females x 100
Number of females paired

Conception index (%) Number of pregnant females x 100
Number of females mated

Gestation index (%) Number of females bearing live pups x 100
Number of pregnant females

Duration of gestation Number of days between confirmation of mating and the beginning of parturition

Percentage live males at First Litter Check Number of live male pups at First Litter Check x 100
Number of live pups at First Litter Check

Percentage live females at First Litter Check Number of live female pups at First Litter Check x 100
Number of live pups at First Litter Check

Percentage of postnatal loss Number of dead pups before planned necropsy x 100
Number of live pups at First Litter Check

Viability index Number of live pups before planned necropsy x 100
Number of pups born alive

Historical control data:
Historical control data for number of living pups at first litter check for rats of the strain and age used in this study: mean of 1083 litters = 11.7; P5 = 7.0; P95 = 15.0.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: reduce body weight gain

Details on maternal toxic effects:
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.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: reduced body weight gain

Details on embryotoxic / teratogenic effects:
At 125 mg/kg, the sex ratio of the pups indicated more female than male pups (65% female pups at 125 mg/kg versus 41% female pups in the control group). As there was no dose-related response, this finding was not attributed to treatment.

The numbers of females with living pups on Day 1 of lactation were 6, 9, 6 and 10 at 0, 30, 125 and 500 mg/kg, respectively. The lower number of litters at 125 mg/kg was considered to be unrelated to treatment with the test substance because the number of litters at 500 mg/kg was normal. For unknown reasons, the number of litters in the control group was also lower than normal. This was considered not to have precluded a meaningful evaluation of the results as explained in Chapter 8 Discussion and Conclusion.

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and the early postnatal pup development endpoints mortality, clinical signs and macroscopy were observed. A toxicologically relevant decrease in pup body weight at Day 4 of lactation was noted at 500 mg/kg.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Gestation

The gestation index and duration of gestation were unaffected by treatment up to 500 mg/kg bw/day.

 

Parturition/maternal care

No signs of difficult or prolonged parturition were noted among the pregnant females.Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.No deficiencies in maternal care were observed.

 

Earlypostnatalpup development

The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs and external macroscopy did not reveal treatment-related findings.

 


 

The mean number of living pups at first litter check was lower (not statistically significantly) at 125 mg/kg than in the control group. There was no dose-related response and the values were within normal limits1. Therefore, this finding was considered not to be related to treatment. 

 

1Historical control data for number of living pups at first litter check for rats of the strain and age used in this study: mean of 1083 litters = 11.7; P5 = 7.0; P95 = 15.0.

 

Mortality

At the first litter check, two pups of one control female (no. 49) were found dead. The control female (no. 41) which was sacrificed because of total litter loss had one pup which was partly cannibalized at first litter check (this pup is not included in the tables in Appendix 1 and 2). During lactation, two pups of the control group, one pup at 30 mg/kg, one pup at 125 mg/kg and one pup at 500 mg/kg went missing. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

 

Clinical signs

Pups that went missing showed no clinical signs. Incidental clinical signs seen in surviving pups consisted of a blue spot on the neck, or scabs on the neck or snout.The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

 

Body weights

At 500 mg/kg mean body weights on Day 4 of lactation (male and female pups) were slightly (about 10%) lower compared to controls. The differences from controls were not statistically significant.

 

Macroscopy

Incidentalmacroscopic findings of pups that were found dead consisted of advanced or beginning autolysis and absence of milk in the stomach in two pups of the control group. Macroscopic findings among surviving pups were limited to scabs in the neck of one pup in the control group. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were thereforeconsidered to be of no toxicological relevance.

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.