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Toxicological information

Toxicity to reproduction

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 04 November 2009 and 18 March 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 6 May 2008 and 20 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: 320 to 355g (male), 184 to 225g (female)
- 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, the 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: Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K., Oxon, UK), ad libitum
- Water: Mains water, ad libitum, was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: twelve days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hr dark / hrs light): 12 hours continuous light and 12 hours darkness
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared twice monthly and stored at approximately 4°C in the dark

VEHICLE
- Justification for use and choice of vehicle: Most suitable
- Concentration in vehicle: 25, 75 and 250 mg/ml.
- Amount of vehicle (if gavage): 4 ml/kg bodyweight
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
On day 15, animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vagina smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. 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) and the males were subsequently returned to their original cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. The male dose groups were killed and examined macroscopically on Day 43. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Duration of treatment / exposure:
Oral administration of the test substance to all rats for a period of up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).
Frequency of treatment:
7 days/week
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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
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. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. 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. 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. The male dose groups were killed and examined macroscopically on Day 43. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then 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. Terminal bodyweights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. For females showing evidence of mating, food consumption was recorded for 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).

FOOD EFFICIENCY:
- Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes

PREGNANCY AND PARTURITION:
- Each female was observed at approximately 08:30, 12:30, and 16:30 hours and around the period of expected parturition. Observations were carried out a approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female: Date of pairing, Date of mating, Date and time of observed start of parturition, and Date and time of observed completion of parturition.

GROSS PATHOLOGY: Yes
- Adult females were killed by intravenous overdose with sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce litter were killed on Day 26 post coitum. 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 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 were stated: Ovaries, Mammary tissue, Pituitary, Uterus/Cervix, Vagina. The tissues from control and high dose group, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination.

REPRODUCTIVE INDICES
- Mating Performance and Fertility: The following parameters were calculated from the individual data during the mating period of the parental generation: i) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating. ii)Fertility Indices: For each group the following were calculated: Mating Index (%) = No.of Animals mated / No. of Animals Paired x 100; Pregnancy Index (%) = No.of Pregnant Females / No. of Animals mated x 100
- Gestation and Parturition Data: The following parameters were calculated from individual data during the gestation and parturition period of the parental generation: i)Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. ii) Parturition Index: The following was calculated for each group: Parturition Index (%) = No. Females Delivering Live Offspring / No. of Pregnant Females x 100
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded: Number of offspring born, number of offspring alive recorded on Days 1 and 4, post partum; Sex of offsprings on Days 1 and 4 post partum, Clinical condition of offspring from birth to Day 5 post partum; Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data). All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS PATHOLOGY
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

OFFSPRING VIABILITY INDICES
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows: % pre–implantation loss= No. of Corpora Lutea - No. of Implantation Sites / No. of Corpora Lutea x 100; % post–implantation loss = No. of Implantation Sites – Total No. of Offspring Born / Number of Implantation Sites x 100; ii) Live Birth and Viability Indices: The following indices were calculated for each litter as follows: Live Birth Index (%) = No. of Offspring alive on Day 1 / No. of Offspring x 100; Viability Index (%) = No. of Offspring alive on Day 4 / No. of Offspring alive on Day 1 x100; iii) Sex Ratio (% males): Sex ratio was calculated for each litter value on Days 1 and 4post partum, using the following formula: No. of Male Offspring / Total No. of Offspring x 100
Statistics:
The following statistical procedures were used for males and females during the pre-mating phase, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data for females during gestation and lactation, and offspring 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.

Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Episodes of increased salivation were evident females treated with 1000 and 300 mg/kg bw/day throughout the treatment period. One female treated with 300 mg/kg bw/day had generalised fur loss between Days 44 and 46. An observation of this nature is commonly observed during the lactation phase and is considered not to be toxicologically significant. A female from this treatment also had a mass from Day 33 onwards. In the absence of a true dose related response these observations were not considered to be related to test material toxicity.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically significant effect on bodyweight development was detected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption was detected in treated females throughout the two week maturation period or during the gestation or lactation phases of the study.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food efficiency was detected in treated females throughout the two week maturation period or during the gestation or lactation phases of the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the organ weights measured. Statistical analysis did not reveal any significant intergroup differences.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected. One female treated with 100 mg/kg/day had a mass observed at necropsy. In the absence of any histology correlates or similar effects present in 300 or 1000 mg/kg bw/day animals the intergroup difference was considered not to be of toxicological importance. A female from the control group showed the liver protruding through the diaphragm. In view of the fact this macroscopic observation was present in a control animal only, it was considered unrelated to treatment. One female treated with 300 mg/kg/day had generalised fur loss at necropsy. Observations of this nature are commonly observed following lactation and are considered not to be toxicologically significant.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic observations were detected.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no significant intergroup differences in gestation lengths and no effects upon parturition for treated females, with all of the females giving birth following 22 to 24 days of gestation. The distribution for treated females was comparable to controls
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects detected in conception rates. One female treated with 300 mg/kg bw/day was non pregnant.
Other effects:
no effects observed
Description (incidence and severity):
The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg/day. There were no treatment related effects on mating performance. The distribution of precoital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). One female treated with 100 mg/kg bw/day did not show any positive evidence of mating but subsequently gave birth to live offspring.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gains were considered to have been unaffected by maternal exposure.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio (percent of males) at birth, on Day 1 and on Day 4 was also unaffected. Litter from females treated with 300 mg/kg/day did show a statistically significant reduction in the percent of males at birth, Day 1 and Day 4. In the absence of a similar effect in litters from females treated with 1000 mg/kg/day the intergroup difference was considered not to be of toxicological importance.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size at Day 1 for treated animals was similar to controls. Mean litter weights were considered to have been unaffected by maternal exposure.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.
External malformations:
no effects observed
Description (incidence and severity):
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.
Details on embryotoxic / teratogenic effects:
The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of age were unaffected by maternal exposure.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The oral administration of the test substance to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related reproductive effects. The NOAEL for maternal and developmental toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

The potential adverse effects of the test material on reproduction including offspring development were studied in an OECD 421 Reproduction/Developmental Toxicity Screening Test following GLP. The test material was administered by gavage to three groups, each of ten male and ten female Wistar rats, for up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Adult males were terminated on Day 43, and all females and surviving offspring on Day 5 post partum. Clinical signs, bodyweight change, 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. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. There were no unscheduled deaths and no clinically observable signs of toxicity detected. No adverse effect on bodyweight change, food consumption or food efficiency was detected for treated animals when compared with controls. No toxicologically significant macroscopic abnormalities were detected, no treatment-related effects were detected in the organ weights measured, and no treatment-related microscopic changes were observed. There were no treatment-related effects on mating, conception rates, and gestation lengths. Of the litters born, litter size, bodyweight gain and litter weight at birth and on day 1 and 4 were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups. In conclusion, the oral administration of the test substance to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related systemic or reproductive effects. The NOAEL for maternal and developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 04 November 2009 and 18 March 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: 320 to 355g (male), 184 to 225g (female)
- 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, the 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: Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K., Oxon, UK), ad libitum
- Water: Mains water, ad libitum, was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: twelve days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hr dark / hrs light): 12 hours continuous light and 12 hours darkness
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared twice monthly and stored at approximately 4°C in the dark

VEHICLE
- Justification for use and choice of vehicle: Most suitable
- Concentration in vehicle: 25, 75 and 250 mg/ml.
- Amount of vehicle (if gavage): 4 ml/kg bodyweight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test material formulations were previously determined. Results showed the formulations to be stable for at least twenty days. Samples of each test material formulation were taken and analysed. The results indicate that the prepared formulations were within ±7% of the nominal concentration.

METHOD
- Samples: The test material formulations were extracted with acetonitrile to give a final, theoretical concentrations of approximately 0.1 mg/ml.
- Standards: Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.
- Procedure: The concentration of test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. HPLC: Agilent Technologies 1200, incorporating autosampler and workstation; Column: Sonoma C18 5µ (250 x 4.6 mm id); Column temp: 40°C; Mobile phase: Acetonitrile; Flow-rate: 1ml/min; UV detector wavelength: 203nm; Injection volume: 25µl; Retention time: ~4.0 mins.
Duration of treatment / exposure:
Oral administration of the test substance to all rats for a period of up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).
Frequency of treatment:
7 days/week
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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of previous toxicity work (Harlan Project Number 1543-0230).
- Mating: On day 15, animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vagina smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. 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) and the males were subsequently returned to their original cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. The male dose groups were killed and examined macroscopically on Day 43. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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 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).

FOOD EFFICIENCY:
- Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose with sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce litter were killed on Day 26 post coitum. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. For 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 were stated: Coagulation gland, Epididymides*, Ovaries, Mammary tissue (females only), Pituitary, Prostate, Seminal vesicles, Testes*, Uterus/Cervix, Vagina (*preserved in Bouin's fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later). The tissues from control and high dose group, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 1000 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Other examinations:
Reproductive performance
- Pregnancy and parturition: Each female was observed at approximately 08:30, 12:30, and 16:30 hours and around the period of expected parturition. Observations were carried out a approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female: Date of pairing, Date of mating, Date and time of observed start of parturition, and Date and time of observed completion of parturition.
- Litter data: On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded: Number of offspring born, number of offspring alive recorded on Days 1 and 4, post partum; Sex of offsprings on Days 1 and 4 post partum, Clinical condition of offspring from birth to Day 5 post partum; Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data).
- Physical development: All live offspring were assessed for surface righting reflex on Day 1 post partum.
 
Reproductive Indices
- Mating Performance and Fertility: The following parameters were calculated from the individual data during the mating period of the parental generation: i) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating. ii)Fertility Indices: For each group the following were calculated: Mating Index (%) = No.of Animals mated / No. of Animals Paired x 100; Pregnancy Index (%) = No.of Pregnant Females / No. of Animals mated x 100
- Gestation and Parturition Data: The following parameters were calculated from individual data during the gestation and parturition period of the parental generation: i)Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. ii) Parturition Index: The following was calculated for each group: Parturition Index (%) = No. Females Delivering Live Offspring / No. of Pregnant Females x 100
- Litter Responses: The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age). i) Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows: % pre–implantation loss= No. of Corpora Lutea - No. of Implantation Sites / No. of Corpora Lutea x 100; % post–implantation loss = No. of Implantation Sites – Total No. of Offspring Born / Number of Implantation Sites x 100; ii) Live Birth and Viability Indices: The following indices were calculated for each litter as follows: Live Birth Index (%) = No. of Offspring alive on Day 1 / No. of Offspring x 100; Viability Index (%) = No. of Offspring alive on Day 4 / No. of Offspring alive on Day 1 x100; iii) Sex Ratio (% males): Sex ratio was calculated for each litter value on Days 1 and 4post partum, using the following formula: No. of Male Offspring / Total No. of Offspring x 100
Statistics:
The following statistical procedures were used for males and females during the pre-mating phase, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data for females during gestation and lactation, and offspring 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.

Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Episodes of increased salivation were evident in males from all treatment groups and in females treated with 1000 and 300 mg/kg bw/day throughout the treatment period. Three males treated with 300 mg/kg bw/day also showed instances of a red/brown stained snout or mouth. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test material formulation and in isolation are considered not to be of toxicological importance. One female treated with 300 mg/kg bw/day had generalised fur loss between Days 44 and 46. An observation of this nature is commonly observed during the lactation phase and is considered not to be toxicologically significant. One male treated with 100 mg/kg bw/day had a mass from Day 36 onwards, was lethargic on Days 33 and 34 and had hunched posture between Days 33 and 39. A female from this treatment also had a mass from Day 33 onwards. In the absence of a true dose related response these observations were not considered to be related to test material toxicity.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effect on bodyweight development was detected. Males treated with 1000 and 300 mg/kg/ bwday showed a statistically significant reduction in bodyweight gain during Week 3. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption or food efficiency was detected in treated males throughout the treatment period. No adverse effect on food consumption or food efficiency was detected in treated females throughout the two week maturation period or during the gestation or lactation phases of the study.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the organ weights measured. Statistical analysis did not reveal any significant intergroup differences.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring: Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.
Adults: No toxicologically significant macroscopic abnormalities were detected. One male and one female treated with 100 mg/kg/day had a mass observed at necropsy. In the absence of any histology correlates or similar effects present in 300 or 1000 mg/kg bw/day animals the intergroup differences were considered not to be of toxicological importance. One control male had a small right testis (left testis not present) and left epididymis at necropsy. A female from the control group showed the liver protruding through the diaphragm. In view of the fact these macroscopic observations were present in control animals only they were considered unrelated to treatment. One female treated with 300 mg/kg/day had generalised fur loss at necropsy. Observations of this nature are commonly observed following lactation and are considered not to be toxicologically significant. One male treated with 1000 mg/kg/day had a mottled appearance of the kidneys at necropsy. In the absence of any histopathological correlates the intergroup difference was considered of no toxicological importance.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic observations were detected.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Mating: There were no treatment related effects on mating performance. The distribution of precoital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). One female treated with 100 mg/kg bw/day did not show any positive evidence of mating but subsequently gave birth to live offspring.
- Fertility: There were no treatment-related effects detected in conception rates. One female treated with 300 mg/kg bw/day was non pregnant.
- Gestation Length: There were no significant intergroup differences in gestation lengths and no effects upon parturition for treated females, with all of the females giving birth following 22 to 24 days of gestation. The distribution for treated females was comparable to controls.
- Litter Response: In total all control females, all females from the 100 mg/kg bw/day dose group and nine females from the 300 and 1000 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. One female treated with 1000 mg/kg bw/day had a total litter loss on Day 2 post partum.
- Offspring Litter Size and Viability: The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day. Subsequent litter size at Day 1 for treated animals was similar to controls. Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls. Sex ratio (percent of males) at birth, on Day 1 and on Day 4 was also unaffected. Litter from females treated with 300 mg/kg bw/day did show a statistically significant reduction in the percent of males at birth, Day 1 and Day 4. In the absence of a similar effect in litters from females treated with 1000 mg/kg bw/day the intergroup difference was considered not to be of toxicological importance.
- Offspring Growth and Development: Mean litter weights and bodyweight gains were considered to have been unaffected by maternal exposure. The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of age were unaffected by maternal exposure.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Conclusions:
The oral administration of the test substance to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related reproductive effects. The NOAEL for systemic and reproductive toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

The potential adverse effects of the test material on reproduction including offspring development were studied in an OECD 421 Reproduction/Developmental Toxicity Screening Test following GLP. The test material was administered by gavage to three groups, each of ten male and ten female Wistar rats, for up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Adult males were terminated on Day 43, and all females and surviving offspring on Day 5 post partum. Clinical signs, bodyweight change, 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. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. There were no unscheduled deaths and no clinically observable signs of toxicity detected. No adverse effect on bodyweight change, food consumption or food efficiency was detected for treated animals when compared with controls. No toxicologically significant macroscopic abnormalities were detected, no treatment-related effects were detected in the organ weights measured, and no treatment-related microscopic changes were observed. There were no treatment-related effects on mating, conception rates, and gestation lengths. Of the litters born, litter size, bodyweight gain and litter weight at birth and on day 1 and 4 were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups. In conclusion, the oral administration of the test substance to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related systemic or reproductive effects. The NOAEL for systemic and reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

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
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: 320 to 355g (male), 184 to 225g (female)
- 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, the 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: Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K., Oxon, UK), ad libitum
- Water: Mains water, ad libitum, was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: twelve days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hr dark / hrs light): 12 hours continuous light and 12 hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared twice monthly and stored at approximately 4°C in the dark

VEHICLE
- Justification for use and choice of vehicle: Most suitable
- Concentration in vehicle: 25, 75 and 250 mg/ml.
- Amount of vehicle (if gavage): 4 ml/kg bodyweight
Details on mating procedure:
On day 15, animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vagina smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. 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) and the males were subsequently returned to their original cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. The male dose groups were killed and examined macroscopically on Day 43. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test material formulations were previously determined. Results showed the formulations to be stable for at least twenty days. Samples of each test material formulation were taken and analysed. The results indicate that the prepared formulations were within ±7% of the nominal concentration.

METHOD
- Samples: The test material formulations were extracted with acetonitrile to give a final, theoretical concentrations of approximately 0.1 mg/ml.
- Standards: Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.
- Procedure: The concentration of test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. HPLC: Agilent Technologies 1200, incorporating autosampler and workstation; Column: Sonoma C18 5µ (250 x 4.6 mm id); Column temp: 40°C; Mobile phase: Acetonitrile; Flow-rate: 1ml/min; UV detector wavelength: 203nm; Injection volume: 25µl; Retention time: ~4.0 mins.
Duration of treatment / exposure:
Oral administration of the test substance to all rats for a period of up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).
Frequency of treatment:
7 days/week
Details on study schedule:
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. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. 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. 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. The male dose groups were killed and examined macroscopically on Day 43. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of previous toxicity work (Harlan Project Number 1543-0230).

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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 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).

FOOD EFFICIENCY:
- Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes

PREGNANCY AND PARTURITION:
- Each female was observed at approximately 08:30, 12:30, and 16:30 hours and around the period of expected parturition. Observations were carried out a approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female: Date of pairing, Date of mating, Date and time of observed start of parturition, and Date and time of observed completion of parturition.
Litter observations:
- Litter data: On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded: Number of offspring born, number of offspring alive recorded on Days 1 and 4, post partum; Sex of offsprings on Days 1 and 4 post partum, Clinical condition of offspring from birth to Day 5 post partum; Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data).
- Physical development: All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose with sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce litter were killed on Day 26 post coitum. 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 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 were stated: Coagulation gland, Epididymides*, Ovaries, Mammary tissue (females only), Pituitary, Prostate, Seminal vesicles, Testes*, Uterus/Cervix, Vagina (*preserved in Bouin's fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later). The tissues from control and high dose group, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 1000 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. All 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 statistical procedures were used for males and females during the pre-mating phase, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data for females during gestation and lactation, and offspring 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.

Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Reproductive indices:
- Mating Performance and Fertility: The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii)Fertility Indices: For each group the following were calculated: Mating Index (%) = No.of Animals mated / No. of Animals Paired x 100; Pregnancy Index (%) = No.of Pregnant Females / No. of Animals mated x 100

- Gestation and Parturition Data: The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i)Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index: The following was calculated for each group: Parturition Index (%) = No. Females Delivering Live Offspring / No. of Pregnant Females x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows: % pre–implantation loss= No. of Corpora Lutea - No. of Implantation Sites / No. of Corpora Lutea x 100; % post–implantation loss = No. of Implantation Sites – Total No. of Offspring Born / Number of Implantation Sites x 100;
ii) Live Birth and Viability Indices: The following indices were calculated for each litter as follows: Live Birth Index (%) = No. of Offspring alive on Day 1 / No. of Offspring x 100; Viability Index (%) = No. of Offspring alive on Day 4 / No. of Offspring alive on Day 1 x100;
iii) Sex Ratio (% males): Sex ratio was calculated for each litter value on Days 1 and 4post partum, using the following formula: No. of Male Offspring / Total No. of Offspring x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Episodes of increased salivation were evident in males from all treatment groups and in females treated with 1000 and 300 mg/kg bw/day throughout the treatment period. Three males treated with 300 mg/kg bw/day also showed instances of a red/brown stained snout or mouth. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test material formulation and in isolation are considered not to be of toxicological importance. One female treated with 300 mg/kg bw/day had generalised fur loss between Days 44 and 46. An observation of this nature is commonly observed during the lactation phase and is considered not to be toxicologically significant. One male treated with 100 mg/kg bw/day had a mass from Day 36 onwards, was lethargic on Days 33 and 34 and had hunched posture between Days 33 and 39. A female from this treatment also had a mass from Day 33 onwards. In the absence of a true dose related response these observations were not considered to be related to test material toxicity.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effect on bodyweight development was detected. Males treated with 1000 and 300 mg/kg/ bwday showed a statistically significant reduction in bodyweight gain during Week 3. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption was detected in treated males throughout the treatment period. No adverse effect on food consumption was detected in treated females throughout the two week maturation period or during the gestation or lactation phases of the study.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food efficiency was detected in treated males throughout the treatment period. No adverse effect on food efficiency was detected in treated females throughout the two week maturation period or during the gestation or lactation phases of the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic observations were detected.

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
- Mating: There were no treatment related effects on mating performance. The distribution of precoital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). One female treated with 100 mg/kg bw/day did not show any positive evidence of mating but subsequently gave birth to live offspring.
- Fertility: There were no treatment-related effects detected in conception rates. One female treated with 300 mg/kg bw/day was non pregnant.
- Gestation Length: There were no significant intergroup differences in gestation lengths and no effects upon parturition for treated females, with all of the females giving birth following 22 to 24 days of gestation. The distribution for treated females was comparable to controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of age were unaffected by maternal exposure.
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg/day. Subsequent litter size at Day 1 for treated animals was similar to controls. Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean litter weights and bodyweight gains were considered to have been unaffected by maternal exposure.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio (percent of males) at birth, on Day 1 and on Day 4 was also unaffected. Litter from females treated with 300 mg/kg/day did show a statistically significant reduction in the percent of males at birth, Day 1 and Day 4. In the absence of a similar effect in litters from females treated with 1000 mg/kg/day the intergroup difference was considered not to be of toxicological importance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

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

Conclusions:
The oral administration of the test substance to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related reproductive effects. The NOAEL for systemic and reproductive toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

The potential adverse effects of the test material on reproduction including offspring development were studied in an OECD 421 Reproduction/Developmental Toxicity Screening Test following GLP. The test material was administered by gavage to three groups, each of ten male and ten female Wistar rats, for up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Adult males were terminated on Day 43, and all females and surviving offspring on Day 5 post partum. 

Clinical signs, bodyweight change, 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. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results on systemic toxicity: There were no unscheduled deaths and no clinically observable signs of toxicity detected. No adverse effect on bodyweight change, food consumption or food efficiency was detected for treated animals when compared with controls. No toxicologically significant macroscopic abnormalities were detected, no treatment-related effects were detected in the organ weights measured, and no treatment-related microscopic changes were observed.

Results on fertility: There were no treatment-related effects on mating, conception rates, and gestation lengths. Of the litters born, litter size, bodyweight gain and litter weight at birth and on day 1 and 4 were comparable to controls.

Results on developmentall toxicity: No clinically observable signs of toxicity were detected for offspring from all treatment groups. In conclusion, the oral administration of the test substance to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related systemic or reproductive effects. The NOAEL for systemic and reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.