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Diss Factsheets

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:
The study was performed between 30 March 2010 and 30 July 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/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Date of GLP Inspection: 15th September 2009. Date of signature on GLP certificate: 26th November 2009.
Limit test:
no

Test material

Constituent 1
Reference substance name:
E0286P/040A
IUPAC Name:
E0286P/040A
Details on test material:
Sponsor's identification: E0286P/040A
Description: amber colured viscous liquid
Batch number: unknown
Label: E0286P/040/04 Irritant
Date received: 11 February 2010
Storage conditions: room temperature in the dark
Expiry date: 20 July 2011

Test animals

Species:
rat
Strain:
other: Wistar Han™:HsdRccHan™:WIST strain rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon

- Age at study initiation: Approx. 12 weeks

- Weight at study initiation: males weighed 333 to 385g, the females weighed 199 to 238g

- Fasting period before study: Animals were not fasted prior to sampling.

- Housing: Initially, all animals were housed in groups of five in polpropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, the non-recovery 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. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.

- Diet: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K., Oxon, UK) was used (ad libitum).

- Water (e.g. ad libitum): ad libitum mains water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Acclimation period: The animals were acclimatised for twelve days


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 21˚C +/- 2˚C

- Humidity (%): 55 +/- 15%

- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.

- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES:
The in-life phaseof the study was conducted between 30 March 2010 (first day of treatment) and 25 May 2010 (final necropsy).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test material was initially prepared at the appropriate concentrations as a solution in Polyethylene Glycol 400 (PEG 400). Thestability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services. Results from the previous study showed the formulations to be stable for at least four hours. Formulations were therefore prepared daily and dosed within this time frame. It was noticed that the formulations at the high dose became very viscous over a very short period of time, resulting in difficulties in administration to the animals. From visual inspection of the test material formulations in PEG 400 and trial formulations prepared in Dried PEG 400, a more suitable dosing formulation was identified using Dried PEG 400 as a vehicle. Dried PEG 400 was therefore employed as the vehicle from Day 24 of the study.


Details on mating procedure:
Non-recovery 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 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) and the males were subsequently returned to their original holding cages (unless required for additional pairing). 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:
The concentration of E0286P/040A in the test material formulations was determined at least weekly by gas chromatography (GC) using an external standard technique.

Samples:
The test material formulations were diluted with tetrahydrofuran to give a final, theoretical test material concentration of approximately 0.1 mg/ml.

Standards:
Standard solutions of test material were prepared in tetrahydrofuran at a nominal concentration of 0.1 mg/ml.

Procedure:
The standard and sample solutions were analysed by GC using the following conditions:

-GC system: Agilent Technologies 5890, incorporating autosampler and workstation
-Column: DB-1 or 2B-1 (30m x 0.32 mm id x 0.25 um film)
-Oven temperaure program: initial 50 °C for 0 mins rate 10 °C/min, rate 50 °C/min; temp 150 °C for 0 mins, rate 50 °C/min; final 325 °C for 10 mins
-Injection temperature: 250 °C
-Flame ionisation detector temperature: 300 °C
- Injection volume 1ul
- Retention period: Profile of peaks at approx. 6.5 mins

Conclusion:

The analytical method was satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study. The change of vehicle from PEG 400 to Dried PEG 400 was not considered to affect the purpose or integrity of the study and did not impact on stability determinations.


Duration of treatment / exposure:
Males dosed for at least 43 days, beginning 14 days prior to mating (Groups 1-4).
Dosing of the females beginning 14 days prior to mating and continuing through mating, up to and including GD 19 (Groups 1-4). Another set of animals (Groups 5 and 6) dosed for a total of 43 days before beginning a 14-day (non-treated) recovery period.
Frequency of treatment:
Non-Recovery Dose Groups:
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.
iii) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post parium, all surviving females and surviving offspring were killed and examined macroscopically.

Recovery Dose Groups:
i) Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii) The males and females were maintained without treatment for a further fourteen days.
iii) After fourteen days of recovery, males and females were killed and examined macroscopically.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 12 weeks of age
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 30; 300; 1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Terminal Groups (Groups 1 to 4): 10 animals per sex per dose
Recovery Groups (Groups 5 and 6): 5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The study was performed according to OECD guidline 422 and was designed to screen for potential adverse effects on reproduction, including offspring development, by repeated oral administration to the Wistar Han™:HsdRccHan™:WIST strain rat for up to fifty-four consecutive days at dose levels of 30, 300 and 1000 mg/kg/day. A control group was dosed with vehicle alone (Polyethylene glycol 400/Dried Polyethylene Glycol 400). Two recovery groups were treated with the high dose level (1000 mg/kg/day) or the vehicle alone for up to forty-two days and then maintained without treatment for a further fourteen days.

The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd. UK Project Number 2184-0016). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material.

Positive control:
Not included

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition, where applicable). During the treatment-free period, recovery animals were observed daily. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: For non-recovery animals, 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 parium. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Not determined
Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals adult non-recovery males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: All surviving non-recovery adult females were killed by intravenous overdose of sodium pentobarbitone followed by
exsanguination on Day 5 post partum.
-Recovery group animals: Recovery group animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 57.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. For all non-recovery 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).

ORGAN WEIGHTS
The epididymides and testes were removed from terminal kill adult males, dissected free from fat and weighed before fixation. In addition, the thyroid, liver and kidneys were also weighed for all surviving adult animals at termination.

HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group,in buffered 10% formalin, except where stated:

Coagulating gland
Epididymides*
Kidneys
Liver
Ovaries
Mammary gland (females only)
Pituitary
Prostate
Seminal vesicles
Testes*
Thyroid
Uterus/Cervix
Vagina

*preserved in Bouin's fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group,in buffered 10% formalin, except where stated:

Coagulating gland
Epididymides*
Kidneys
Liver
Ovaries
Mammary gland (females only)
Pituitary
Prostate
Seminal vesicles
Testes*
Thyroid
Uterus/Cervix
Vagina

*preserved in Bouin's fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
Statistics:
See below
Reproductive indices:
See below
Offspring viability indices:
See below

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

See below

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

Details on results (F1)

See below

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

RESULTS

ADULT RESPONCES

-Mortality. There were no unscheduled deaths considered to be attributable to test material toxicity.

-Clinical Observations. Clinical observations for surviving animals mainly consisted of post-dose increased salivation and noisy respiration for animals of either sex treated at all dose levels. Severe clinical signs were observed for one male treated with 300 mg/kg/day on Day 2 resulting in the termination of this animal. One male treated with 1000 mg/kg/day showed respiratory pattern changes and lethargy on Day 3, and this animal was also terminated. One high dose female showed severe clinical signs resulting in the termination of this animal on Day 33.

-Bodyweight. Non-recovery 1 000 mg/kg/day males showed lower bodyweight gains during the treatment period when compared to controls. Slight reductions in bodyweight gains were also observed for recovery group animals treated at 1000 mg/kg/day during the first week of treatment, although regression was evident thereafter. No adverse effects on bodyweight change were detected during the gestation and lactation phases of the study for non-recovery females.

-Food Consumption and Food Efficiency. A slight reduction in dietary intake and food efficiency was evident for non-recovery 1000 mg/kg/day males during the treatment period when compared controls. This reduction was also evident for non-recovery females and animals allocated to the recovery groups during the first week of the treatment period, with regression observed thereafter. No adverse effects on dietary intake were evident during gestation and lactation for nonrecovery group females.

-Water Consumption. Daily visual inspection of water bottles did not reveal any significant intergroup differences.

Reproductive Screening:

-Mating. No treatment-related effects were detected in mating performance.

-Fertility. No treatment-related effects on fertility were detected.

-Gestation Lengths. No treatment-related effects were detected in the length of gestation between control and treated groups.

LITTER RESPONCES:

-Offspring Litter Size, Sex Ratio and Viability. No treatment-related effects were detected.

-Offspring Growth and Development. No treatment-related effects were detected.

PATHOLOGY:

-Necropsy. Macroscopic findings for terminal kill animals consisted of enlarged livers, and a mottled appearance and pallor of the kidneys for males treated with 1000 mg/kg/day, with the effect extending into males from the 300 and 30 mg/kg/day dose groups. A mottled appearance of the kidneys was also evident for one recovery 1000 mg/kg/day male after the fourteen day treatment-free period. No treatment-related macroscopic changes of the kidneys were recorded for females. The interim death animals displayed gaseous distension of the gastro-intestinal tract.

-Organ Weights. No treatment-related effects were evident in epididymis or testis weights from treated males compared to controls. Increases in absolute and bodyweight-relative liver and kidney weights were evident for non-recovery animals of either sex treated with 1000 mg/kg/day when compared to controls. Non-recovery 1 000 mg/kg/day males also showed an increase in absolute and bodyweight-relative thyroid weights. These increases extended into the male 300 mg/kg/day dose group. No such elevations were evident following the fourteen day treatment-free in recovery 1 000 mg/kg/day animals of either sex.

-Histopathology. No treatment-related microscopic findings were detected in the reproductive organs. The following treatment-related microscopic findings were detected:

LIVER: Centrilobular/diffuse hepatocellular hypertrophy was observed in all males and one female treated with 1000 mg/kg/day and two males treated with 300 mg/kg/day, but was not present following the fourteen day recovery period.

KIDNEYS: An increased incidence and severity of tubular basophilia and tubular epithelial hyaline droplets/granules were observed for males treated with 1000 mg/kg/day. Tubular degeneration, tubular simple dilation and tubular epithelial hypertrophy were evident for males treated with 1000 mg/kg/day, and were still evident following the fourteen day treatment-free period. No such observations were evident for females.

THYROID GLANDS: An increased incidence of follicular cell hypertrophy was noted in males from the 1000 mg/kg/day dose group, but not for females. Almost complete regression was evident following the recovery period

Applicant's summary and conclusion

Conclusions:
The oral administration of E0286P/040A to rats by gavage at a maximum dose level of 1000 mg/kg/day resulted in treatment-related effects, which were consistent with those previously observed in a twenty-eight day study conducted at this Test Facility (Project Number 2184-0016). Effects seen in the liver and thyroid gland resolved following a 14 day non-treatment recovery period.

No treatment-related effects were detected for reproductive performance, hence the 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.
Executive summary:

The study was performed to screen for potential adverse effects of E0286P/040A on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study meets the requirements of the OECD Guidelines for Testing of Chemicals No. 421 "Reproduction/Developmental Toxicity Screening Test" (adopted 27 July 1995).

The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400/Dried Polyethylene Glycol 400). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

The oral administration of E0286P/040A to rats by gavage at a maximum dose level of 1000 mg/kg/day resulted in treatment-related effects, which were consistent with those previously observed in a twenty-eight day study conducted at this Test Facility (Project Number 2184-0016). Effects seen in the liver and thyroid gland resolved following a 14 day non-treatment recovery period. No treatment-related effects were detected for reproductive performance, hence the 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day

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