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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
of 1996
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: 10 to 11 weeks.
- Weight at treatment start: Males: minimum 321 g, maximum 381 g,
Females: minimum 209 g, maximum 264 g.
- Housing Inside a barriered rodent facility:
all animals pre-mating + toxicity subgroup: In groups up to 5 by sex in solid floor polycarbonate cages.
during mating (1 male+1 female/cage): In RB3 modified propylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after mating: In groups up to 5 by sex in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during mating or post gestation day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Toxicity subgroup animals overnight before blood sampling for hematology/clinical blood chemistry.
- Water (ad libitum): Pottable drinking water from the public supply.
- Acclimation period: 13 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and wood bedding did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity subgroup females: Five weeks
Duration of test, reproductive subgroup females: From 14 days prior to mating to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
Deviations from these target ranges were not evident.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 2 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest body weight. Litter animals were not dosed.

CONCENTRATION OF TEST MATERIAL IN VEHICLE

Group Concentration of Test Substance Treatment Volume Dose Level of Test Substance
(mg/mL) (mL/kg bw/day) (mg/kg bw/day)
Vehicle Control 0 2 0
Low Dose 50 2 100
Mid Dose 150 2 300
High Dose 500 2 1000

Details on mating procedure:
- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- Proof of pregnancy: Formation of copulation plug or sperm in vaginal smear referred to as day 0 of gestation.
(During cohabitation, females were checked every morning for pregnancy).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical analysis of test material formulations by high performance liquid chromatography (HPLC/UV) using an external standard technique.
- Concentrations (verified at first and last treatment week) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the prepared formulations were 102% to 108% of the corresponding nominal concentration.
Duration of treatment / exposure:
- Treatment period, males & toxicity subgroup females: Daily, for five consecutive weeks
- Treatment period, reproductive subgroup females: Ca. 6 to 8 weeks (from 14 days before mating to day 7 of lactation)
Frequency of treatment:
Daily, 7 days/week (during parturition, dosing omitted as appropriate)
Details on study schedule:
- Age at mating of the mated animals in the study: 12 to 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Toxicity subgroups: 5 males* / 5 females
Reproductive subgroups: 5 males* / 10 females
* All males were killed at the same time (Week 6) after pairing with reproductive subgroup females
Control animals:
yes, concurrent vehicle
Details on study design:
This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals and reproductive subgroup animals, whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant). The high dose level of 1000 mg/kg/day was considered to be the maximum practical dose level for the present OECD 422 study and was therefore chosen as the high dose level.
Positive control:
Not included in the study.

Examinations

Parental animals: Observations and examinations:
Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery: During treatment week 5 (before dosing) on all toxicity subgroup animals (5 parental males/group inclusively)*.
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Subgr. Females: Weekly for pre-mating and mating period; on gestation days 0, 7, 14, 20; on lactation days 1, 4 & 7.
- Food consumption, all animals: Weekly for pre-mating period.
Food cons., Repro. Subgr. Females: During gestation for days 0-7, 7-14, 14-20, during lactation for days 1-4 & 4-7.
- Water consumption: Daily (based on visual observation inidicative of overt water intake).
- Hematology: During treatment week 5 after functional observation battery*
- Blood (plasma) chemistry: During treatment week 5 after functional observation battery*

* Examinations confined to toxicity subgroup animals are marked above with an asterisk*
and are detailed in the separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined, oral, rat_HLS_Proj_GAH0008"

Explanatory note
This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals and reproductive subgroup animals whereby 5 of the 10 F0 males (used for mating) per dose group formed the toxicity male subgroups.
Oestrous cyclicity (parental animals):
Frequency of vaginal estrus was determined by examination of vaginal smears taken daily and checking for copulation plugs daily from all reproductive subgroup females from the beginning of the treatment period to the day of confirmed copulation.
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus
Sperm parameters (parental animals):
Parameters examined in male parental animals:
- testis weight,
- epididymis weight
- detailed qualitative histopathology examination of the testes taking into account the tubular stages of the spermatogenic cycle. This was to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS: Not performed. The study ended on lactation day 7.

LITTER PARAMETERS EXAMINED
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Total litter size on day 1 of age and mortality/live litter size on each day until 7 days after littering.
- Sex ratio expressed as percentage males  (Total No. of male pups in litter/Total No. of offspring in litter) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- Clinical signs, recorded daily
- Body weight of live pups (on days 1, 4 and 7 after littering) and weight change from days 1-4 and 1-7.



Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females: Killed on Day 7 post partum. 1 female without viable litter was killed on Day 25 after mating.

Gross pathology
- all rats: Full macroscopic examination  with tissue collection.
- reproduction subgroup females: Recording of the number of uterine implatation sites.

Organs Weights:
- toxicity subgroup animals: Adrenals, brain, epididymides*, heart, kidneys, liver, ovaries*, pituitary, prostate*, seminal vesicles
& coagulation gland*, spleen, testes*, thymus, thyroid with parathyroids, uterus with cervix and oviducts*.
- reproductive subgroup animals: * These organs were also weighed for adult/parental reproductive subgroup animals.
Histopathology:
- reproductive subgroup animals: The following organs were microscopically examined for the control and 1000 mg/kg bw/day groups:
Epididymides, testes, ovaries, uterus (with cervix & oviducts), vagina, mammary area (caudal), seminal vesicle &
coagulation gland, prostate.
- toxicity subgroup animals: For the control and 1000 mg/kg bw/day groups (5 adult/parental males per group inclusive):
Brain, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes,
epididymides, ovaries, lung, trachea, esophagus, stomach, duodenum, jejunum, ileum, caecum, rectum,
colon, Peyer's patch, lymph node (left axillary, mesenteric), urinary bladder, uterus (with cervix & oviducts),
vagina, spinal cord, sciatic nerve, mammary area (caudal),  sternum with marrow, seminal vesicle &
coagulation gland, prostate.
In addition, the kidneys were also examined for the 100 and 300 mg/kg bw/day toxicity subgroups.



Postmortem examinations (offspring):
Pup survivors were killed on Day 7 post partum.
Full macroscopic examination of decedent and surviving pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups could not be examined).
Statistics:
As detailed in Endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined, oral, rat_HLS_Proj_GAH0008"
Reproductive indices:
- No. of animals mating (evidence of successful copulation, i.e. spermatozoa in vaginal smears and/or copulation plugs)
- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Pre-coital interval (pairing days until detection of mating)
- No. of animals achieving pregnancy
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals pairing) x 100
- Gestation length (time elapsing between detection of mating and commencement of parturition
- No. of living pregnant females
- For further reproductive parameters, see also the above section "Litter observations" and section "Offspring viability indices" below.
Offspring viability indices:
- Post-implantation survival index  (Total no. of pups born/Total no. of uterine implantation sites) x 100
- Live birth index (No. of live pups on day 1 after littering/Total no. of pups born) x 100
- Viability index (No. of live pups on day 4 after littering /No. of live pups on day 1 after littering) x 100
- Lactation index (No. of live pups on day 7 after littering /No. of live pups on day 1 after littering) x 100
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
in kidneys at 1000 mg/kg/day: Slight increase in incidence and/or severity of tubular casts in both sexes and in cortical tubular basophilia in females. Examination only in 5 toxicity subgroup females/group and 5 F0 males/group, not examined in F0 females
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
At 1000 mg/kg/day, 1 pair failed to produce a litter. Macroscopically no evidence of any implantation sites in the affected female, microscopically no changes in the reproductive tract in both F0 animals. This was not attributed to the test substance.

Details on results (P0)

Mortality:
- No deaths occurred in all F0 males or females during the study.

Water consumption:
- No overt effects, based on daily visual observation.

Histopathology:
Further details on renal histopathology findings in F0 animals of the present study are given in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined, oral, rat_HLS_Proj_GAH0008", e.g. in Table 1 and the commenting text underneath this table.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: see 'Remark'

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
mortality of pups was observed in all dose groups and the control group within the normal range of rats of this strain.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
until postnatal day 7, the day of scheduled sacrifice of the pups.

Details on results (F1)

No treatment-related abnormality was observed.

No abnormality in sex ratio.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
other: F1 generation was not dosed directly but may have been exposed via milk from dosed dams.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
other: NOAEL = highest dose tested.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion