Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 June 2010 to 28 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/oor minor methodological deficiencies which do not affect the quality of relevant results.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Remarks:
(Date of inspection: 15 September 2009 Date of Signature: 26 November 2009)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DVS005U

- Physical state: Clear colourless viscous liquid.

- Analytical purity: 100%

- Lot/batch No.: MW9F23A901

- Expiration date of the lot/batch: 15 January 2011

- Stability under test conditions: The formulations to be stable for at least twenty days.

- Storage condition of test material: 4°C in the dark, under nitrogen.

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, United Kingdom.

- Age at treatment: Approximately twelve weeks old.

- Weight at study initiation: (male) 272 - 322 g; (female) 195 - 224 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.
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: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK), ad libitum.

- Water: Mains drinking water, ad libitum.

- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C.

- 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: Day 0 To: Day 43 (male); Day 5 post partum (female).

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Formulations were prepared weekly and stored at approximately +4ºC under nitrogen in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was stable for at least 20 days in arachis oil BP and the prepared formulations were within ± 6% of the nominal concentration.

- Concentration in vehicle: 25, 75 and 250 mg/ml

- Amount of vehicle (if gavage): 4 ml/kg

Details on mating procedure:
- M/F ratio per cage: 1:1

- Length of cohabitation: 14 days.

- Proof of mating: The presence of sperm within the vaginal smear and/or vaginal plug in situ.

- Further matings after two unsuccessful attempts: no

- After successful mating each pregnant female was caged (how): Mated 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:
Method; The concentration of DVS005u (aka Weston 705) in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Samples; The test item formulations were diluted with acetonitrile to give a final, theoretical test item concentration of approximately 0.1 mg/ml.

Standards; Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml. The low dose formulations (25 mg/ml) were analysed with standard solutions containing the equivalent amount of vehicle as the sample solutions.

Procedure; The standard and sample solutions were analysed by HPLC.

Homogeneity determinations; The test material formulations were assessed visually.

Stability determinations; The test material formulations were sampled and analysed initially and then after storage at approximately 4ºC in the dark for 20 days.

Verification of test material formulation concentrations; The test material formulations were sampled and analysed within 4 days of preparation.

Results; Measured concentration found was in the range of 94 – 102% of nominal concentrations after 20 days. The results showed the formulation to be stable for at least 20 days.
Duration of treatment / exposure:
Day 0 To: Day 42 (male); Day 4 post partum (female).
Frequency of treatment:
Once daily.
Details on study schedule:
- Age at mating of the mated animals in the study: At least 14 weeks of age.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
10 animals per sex per dose and the control group.
Control animals:
yes, concurrent vehicle
Details on study design:
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, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days and smearing commenced.

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) On Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

- Dose selection rationale: The dose levels were chosen based on the results of the 90 day oral repeated dose toxicity in rats (see Section 7.5.1).
Positive control:
No.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 and public holidays (except for females during parturition where applicable).

- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.


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

FOOD CONSUMPTION: 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 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 post partum).

FOOD EFFICIENCY: Yes
Weekly food efficiency (body weight 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: Yes
Water intake was observed daily by visual inspection of water bottles for any overt changes.

PREGNANCY AND PARTURITION: Yes
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
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 vaginal 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).
Sperm parameters (parental animals):
Parameters examined in parental animals: testes weight, epididymis weight, prostate weight and the presence of sperm within the vaginal smears.
Litter observations:
STANDARDISATION OF LITTERS: Not applicable for this test methodology.

PARAMETERS EXAMINED
The following parameters were examined:
number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on Days 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum and individual offspring weights on Days 1 and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS: Yes
[Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.

- Maternal animals: Adult females were killed by intravenous overdose of a sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY
All adult animals 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.

ORGAN WEIGHTS
The epididymides and testes are removed from terminal kill males, dissected free from fat and weighed before fixation.


HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin.
coagulating gland, epididymides, ovaries, mammary gland, pituitary, prostate, seminal vesicles, testes, uterus/cervix and vagina.
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.
Statistics:
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.
Probability values (P) were calculated as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
p = 0.05 (not significant)
Reproductive indices:
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 (%) = (Number of animals mated÷Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females÷Number of animals mated) x 100

Gestation and Parturition Data

The following parameters were calculated for 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 (%) = (Number of females delivering live offspring÷Number 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 = [(Number of corpora lutea - Number of implantation site)÷Number of corpora lutea] x 100

% post – implantation loss =[(Number of implantation sites - Total number of offspring born)÷ Number of implantation sites)] x 100

Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1÷Number of offspring born) x 100

Viability Index 1 (%) = (Number of offspring alive on Day 4÷Number of offspring alive on Day 1) x 100

iii) Sex Ratio (% males)

Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:

(Number of male offspring ÷ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
There were no unscheduled deaths during the study.


CLINICAL SIGNS
There were no clinical signs of toxicity detected.
One male treated with 1000 mg/kg/day showed increased salivation immediately after dosing and up to one hour after dosing on Day 25 only. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation is considered not to be of toxicological importance.


BODY WEIGHT
No adverse effects on body weight development were detected for all treated animals when compared to controls.
Statistically significant increase in body weight gain was detected during week 6 in males treated with 1000 mg/kg/day. This intergroup difference is considered not to be an adverse toxicological effect.


FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse effect on food consumption was detected for males during the treatment period, or for females during the pre-mating, gestation or lactation phases of the study. Food efficiency (the ratio of body weight gain to dietary intake) was not affected for males throughout the treatment period, or for females during the pre-mating phase.


WATER CONSUMPTION
Daily visual inspection of water bottles did not reveal any significant intergroup differences between control and treated animals.


MATING
There were no treatment-related effects on mating performance.
The majority of animals mated successfully within four days of pairing. Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences.


FERTILITY
There were no treatment-related effects on conception rates.
One female treated with 1000 mg/kg/day and one female treated with 100 mg/kg/day failed to achieve pregnancy following evidence of mating. This finding is occasionally observed in reproductive studies of this type, and in isolation is considered not to represent a toxic effect on fertility.


GESTATION PERIOD
No treatment-related effects were detected for the length of gestation between control and treated groups.
The length of gestation ranged between 22 and 23 ½ days for control and test animals. Statistical analysis of the data did not reveal any significant intergroup differences.


ORGAN WEIGHTS
There were no toxicologically significant effects detected in the organ weights measured.
Statistically significant reductions in testes weight (P < 0.05), both absolute and relative to terminal body weight, was evident in males treated with 1000 mg/kg/day. In the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological significance.


GROSS PATHOLOGY
There were no toxicologically significant macroscopic abnormalities detected.
One male treated with 1000 mg/kg/day had a small prostate and small seminal vesicles at necropsy. At 100 mg/kg/day, two males had small epididymides and small testes at necropsy. In the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological significance.


HISTOPATHOLOGY
No treatment-related microscopic findings were evident.
All gross lesions recorded were considered to be within the range of normal background alterations which may be recorded in animals of this strain and age.
One female treated with 1000 mg/kg/day and one female treated with 100 mg/kg/day were assumed not pregnant during necropsy. The uterus of these animals showed no implantation sites or decidual reaction, vagina and mammary gland showed no pregnancy specific changes. The mated males were without pathological findings.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
(reproductive and development toxicity)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The oral administration of the test material did not result in any treatment-related effects in treated animals at any doses tested.
Remarks on result:
other: Generation: P & F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

In total ten females from control, and 300 mg/kg/day dose groups, and nine females treated with either 100 or 1000 mg/kg/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

LITTER SIZE AND VIABILITY
No significant differences in litter size, viability or sex ratio were evident for offspring from treated litters when compared to those from the controls.
Statistical analysis of the data did not reveal any significant intergroup differences, hence minor intergroup differences were considered to have arisen incidentally and were of no toxicological importance.


CLINICAL SIGNS
No clinically observable signs of toxicity were detected for offspring from treated litters. The clinical signs observed were of low incidence and findings commonly observed in reproductive studies of this type and were unrelated to test item toxicity. Surface righting was not affected at any treatment level.


BODY WEIGHT
No treatment-related effects on litter weight were evident in the treated groups when compared to controls.


GROSS PATHOLOGY
No treatment-related macroscopic findings were detected for offspring.
The incidental findings observed in the offspring were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.





Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of DVS005u (aka Weston 705) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg/day, did not result in any treatment-related effects in treated animals.
The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

Introduction.The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study meets the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to meet the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™: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 100, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, body weight 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.

Adult males were terminated on Day 43, and all females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results.

Adult Responses:

Mortality.There were no unscheduled deaths during the study.

Clinical Observations.There were no clinical signs of toxicity detected.

Body Weight.No adverse effects on body weight development were detected.

Food Consumption and Food Efficiency.No adverse effects on food intake or food efficiency were detected.

Water Consumption.No intergroup differences were detected.

Reproductive Screening:

Mating.There were no treatment-related effects on mating performance.

Fertility.There were no treatment-related effects on conception rates.

Gestation Lengths.There were no differences in gestation lengths. The distribution for treated females were comparable to controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.No significant differences in corpora lutea, implantation sites, or pre- and post- implantation losses were detected for females from treated groups when compared to controls. Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Offspring Growth and Development. No differences in litter weights, offspring weights or surface righting were detected for litters from treated groups when compared to control litters.

Offspring Observations.No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology:

Necropsy.There were no treatment-related macroscopic abnormalities detected.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.There were no treatment-related microscopic effects detected.

Conclusion.The oral administration of DVS005u (aka Weston 705) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg/day, resulted in no treatment-related effects in all treated animals.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was therefore considered to be 1000 mg/kg/day.