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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
Study period:
From July 24, 2009 to December 7, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (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:
liquid
Details on test material:
- Physical state: pale amber liquid
- Analytical purity: 31.8%
- Lot/batch No.: PA19100368
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 12 weeks old
- Weight at study initiation: males: 305-358 g; females: 189-219 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK); ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: September 15, 2009 To: November 8, 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test material formulations were determined. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

Details on mating procedure:
- M/F ratio per cage: on Day 15, all animals were paired on a 1 male:1 female basis within each dose group
- Length of cohabitation: Maximum of 14 days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- After successful mating each pregnant female was caged (how): Females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test material formulation were taken and analysed for concentration. The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 3 mg/ml. Standard solutions of test material were prepared in water at a nominal concentration of 3 mg/ml.

The concentration was determined by high performance liquid chromatography (HPLC) using an external standard technique.
HPLC : Agilent Technologies 1100 or 1200, incorporating autosampler and workstation
Column : Gemini 3µ C18 (100 x 4.6 mm id)
Column temp : 40 °C
Mobile phase : Eluent A:acetonitrile with 0.1% trifluoroacetic acid; Eluent B:water with 0.1% trifluoroacetic acid
Flow-rate : 1 ml/min
UV detector wavelength: 205 nm
Injection volume : 25 µl
Retention time : Profile of peaks from ~ 1.4 to 10.5 mins

The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.
Duration of treatment / exposure:
54 days
Frequency of treatment:
Daily
Details on study schedule:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
actual ingested
Dose / conc.:
15 mg/kg bw/day
Remarks:
actual ingested, incorporating a correction factor for 31.8% purity
Dose / conc.:
40 mg/kg bw/day
Remarks:
actual ingested, incorporating a correction factor for 31.8% purity
Dose / conc.:
100 mg/kg bw/day
Remarks:
actual ingested, incorporating a correction factor for 31.8% purity
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14¿20. For females with live litters, food consumption was recorded during the lactation period (Days 1 ¿ 4).

WATER CONSUMPTION AND COMPOUND INTAKE:
Water intake was observed daily by visual inspection of water bottles for any overt changes. Inter-group differences did not indicate any need for more formal gravimetric measurements.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Days 1 and 4 post partum

All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy were killed after Day 26 post coitum.

GROSS NECROPSY
All adult animals including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where indicated:
Coagulating gland, Prostate, Epididymides, Seminal vesicles, Ovaries, Testes, Mammary tissue, Uterus/Cervix, Pituitary, Vagina.
The tissues from control and 100 mg/kg/day dose group animals, and any animals which failed to achieve a pregnancy, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from control and 100 mg/kg/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:
The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change, Food consumption for females during gestation and lactation, Litter data, Sex ratio, Implantation losses and viability indices, Offspring bodyweight and bodyweight change, Offspring surface righting, Adult absolute and bodyweight relative organ weights.

Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene¿s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett¿s test. Where Levene¿s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ¿U¿ test. The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

In addition, histopathological findings were analysed (excluding non-mated females and females not producing a pregnancy/litter) using the following methods: Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Reproductive indices:
- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices: mating index (%) and pregnancy index (%)
- Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition index
Offspring viability indices:
- Implantation losses (%)
- Live birth and viability indices
- Sex ratio (% males)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant findings were observed throughout the study. Sporadic episodes of increased salivation were evident in animals of either sex treated with 100 mg/kg/day, one male from this treatment group also had red/brown staining around the mouth. One 40 mg/kg/day female had noisy respiration. Generalised fur loss was detected in one 15 mg/kg/day and one control female. Increased salivation is often recorded following the oral administration of an unpleasant tasting test material formulation and is considered not to be indicative of systemic toxicity. The remaining observations are considered incidental and not to represent an adverse effect of treatment. No such effects were detected in the 40 or 15 mg/kg/day males.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically significant effect on bodyweight development was detected for treated animals, in comparison to controls. A slight but statistically significant increase in bodyweight gain (P<0.05) was evident in the 40 and 100 mg/kg/day males during Weeks 5 and 6. This increase was also seen in the 15 mg/kg/day males during Week 6 only. There was no adverse effect on overall bodyweight gain between the treated and control animals. In the absence of an effect on dietary intake or supporting histopathological correlates this finding is considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was monitored daily by visual inspection of the water bottles. There was no adverse effect on water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No adverse effect on organ weight measurement was detected for treated animals, in comparison to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were detected in the treated or control animals at terminal kill.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No adverse effect on mating performance was detected. All animals from the control and treated groups mated within four days of pairing with the exception of one 15 mg/kg/day female which mated after thirteen days of pairing.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
for systemic toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Remarks:
for reproductive toxicity
Effect level:
100 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were detected
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effect on total litter weights, offspring bodyweight development or surface righting reflex was detected in treated animals, in comparison to controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At terminal kill, one female treated with 15 mg/kg/day and one treated with 100 mg/kg/day produced a litter with one pup which showed a dark left testis. One 40 mg/kg/day female produced a litter with one pup which showed a dark right testis. There were no abnormalities detected in the remaining animals. The incidental signs detected were considered to be of no toxicological importance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
VIABILITY (OFFSPRING)
There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls; and pre-implantation and postimplantation losses from treated animals were comparable to controls. Furthermore, no obvious effect on litter size, sex ratio and offspring viability were detected for treated animals, in comparison to controls.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg/day, did not result in any treatment-related effects. The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity).
Executive summary:

The study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guideline No. 421 Reproduction/Developmental Toxicity Screening Test. The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han:HsdRccHan:WIST strain rats, for up to fifty-four consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 15, 40 and 100 mg/kg/day (incorporating a correction factor for 31.8% purity). A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all surviving females and offspring on Day 5 post partum. Any females that failed to achieve pregnancy were killed after Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg/day, did not result in any treatment-related effects.

The 'No Observed Effect Level' (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity).