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Toxicological information

Toxicity to reproduction

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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:
30 November 2009 - .....................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
deviations to study plan but not to guideline
Qualifier:
according to guideline
Guideline:
other: US EPA guideline OPPTS 870.3650, July 2000
Deviations:
no
Remarks:
idem above
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmethacrylate synthesis
IUPAC Name:
Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmethacrylate synthesis
Details on test material:
- Physical state: brown black liquid
- Lot/batch No.: 01/07/09
- Expiration date of the lot/batch: 01/07/2011
- Storage conditions of test material: at room temperature
- Purity/Impurities: not applicable (complex composition)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, l'Arbresle, France
- Age at study initiation: 11 weeks old for the males and 10 weeks old for the females
- Weight at study initiation: 434 g (range: 406 g to 478 g) for the males and 251 g (range: 231 g to 287 g) for the females
- Housing: The animals were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing
autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
Towards the end of gestation and during lactation with their litter, the females were individually housed in polycarbonate cages
43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Autoclaved wood shavings (SICSA, Alfortville, France) were
provided as nesting material, a few days before delivery and during the lactation period.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 9 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (7:00 - 19:00)

IN-LIFE DATES: From: 24 November 2009 To: 05 March 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The sampling of the test item (method supplied by the Sponsor) was performed as follows:
- the container was heated at 80°C for 60 minutes in a water bath and under magnetic stirring until complete dissolution of particles,
- the absence of crystallized particles was checked,
- after complete dissolution of crystallized particles, the appropriate amount of test item was taken into brown flasks and stored at room temperature until use.
The test item was administered as a solution in the vehicle. Before use (weighing, sampling or dilution for the study), each sample of test item was heated and stirred at the temperature which allowed dissolution of crystallized particles until obtaining a solution.
Fresh dosage form preparations were made on the morning of each administration, then the dosage forms were stored at room temperature, and
delivered to the study room in brown flasks.

VEHICLE
- Concentrations in vehicle: 12.5, 50 or 200 of test item/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: nights
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Towards the end of gestation and during lactation with their litter, the females were
individidually housed in polycarbonate cages (43.0 cm x 21.5 cm x 20.0 cm) containing autoclaved sawdust (DICSA, Alfortville, France). Autoclaved
wood shavings were provided as nestinig material, a few days before delivery and during the lactation period.
- Any other deviations from standard protocol: no.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Ionic Chromatography analytical method for the determination of test item in dosage form samples was provided by the Sponsor and this method wa validated at CIT prior to dosage form analysis.

There were no results available for week 1 due to a failure in the analytical sequences for the first and second analyses.
The test item concentrations in the administered dosage forms analyzed in weeks 2, 4, 5 and 6 of treatment remained within the acceptable range
of -9% to -4% of variation compared to the nominal values, except for the value of group 3 in week 4 (43.5 mg/mL, -13%).

Stability: not assessed (complex substance), preparation was done before each administration
Homogeneity: not required (dosage form was a solution)
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing,
- during the pairing period (2 weeks),
- until sacrifice (at least 5 weeks in total).

in the females:
- 2 weeks before pairing,
- during the mating period (2 weeks),
- during gestation,
- during lactation until day 4 post-partum inclusive.

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once daily.
Details on study schedule:
Age at mating of the mated animals in the study: approximately 12 (for the females) or 13 (for the males) weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 250 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous 4-week toxicity study
by oral route in rats (CIT/Study No. 36056 TSR). The test item was administered to three groups of five male and five female Sprague-Dawley rats at
dose-levels of 62.5, 250 or 1000 mg/kg/day under a dose-volume of 5 mL/kg/day. A control group was treated with the vehicle under the same
experimental conditions. In this study, the only in-life findings recorded were isolated and transient clinical signs (1/5 males and 1/5 females given
1000 mg/kg/day displayed reflux at dosing on day 7, and 1/5 females given 250 mg/kg/day had loud breathing on day 16). Neither body weight nor food consumption were affected. There were no treatment-related differences in any of the hematological, blood biochemical or urinary parameters. No organ weight differences were recorded between control and test item-treated animals and there were no remarkable necropsy findings (no
microscopic findings were available at the time of writing of the rationale for dose selection).
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Mortality and morbidity:
- Time schedule: once a day

CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 p.c.
(post-coïtum) and days 1 and 5 p.p. (post partum).

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the
pairing period.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of
pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 p.p.

During the pairing period, the food consumption was measured for neither males nor females.
Food consumption of males was measured after the pairing period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the
females are mated.
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, clinical signs, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

No evaluation of sexual maturation.
Postmortem examinations (parental animals):
A complete macroscopic post-mortem examination was performed on all parent animals. This included examination of the external surfaces, all
orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 5 post partum.

SACRIFICE
- Males were sacrificed after the end of the mating period (at least 5 weeks of treatment in total), females on day 5 p.p.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table below were prepared for microscopic examination and weighed, respectively.
A microscopic examination was performed on:
- all tissues listed in the tissue procedure table in the males and females of the control and high dose groups (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period.
Postmortem examinations (offspring):
Pup examinations
Pups found dead and pups sacrificed on day 5 post-partum were carefully examined externally for gross external abnormalities and a macroscopic
examination was performed. No tissues were preserved.
SACRIFICE
- The offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations, macroscopic

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

HISTOPATHOLOGY / ORGAN WEIGTHS: no
Reproductive indices:
 pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

 post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

 mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

 fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

 gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
 live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

 viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality: none.

Clinical signs
During the pre-mating period, soft feces was noted in one male given 250 mg/kg/day (U22886) or 1000 mg/kg/day (U22895) from day 15 to
day 17, and one other male (U22891) treated at 1000 mg/kg/day showed soiled urogenital area on day 19. Since these signs were recorded
transiently in isolated animals, with incidences not clearly dose-related, and were not observed in any animals from the 4-week toxicity study
carried out at the same dose-levels (CIT/study No. 36056 TSR), a relationship to treatment with the test item was considered to be unlikely.

No other relevant clinical sign was noted during the study in any male or female during premating, gestation or lactation periods, since the few
signs recorded during the study did not show evidence of dose relationship and were observed with low incidence.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight
Males given 250 or 1000 mg/kg/day gained less weight than the controls during the 2 weeks of the premating period (-11% and -48%,
respectively). These differences achieved statistical significance in males given the high-dose only and were mostly due to 4/10 animals which lost weight during week 2, including male U22895 which experienced transient soft feces.
Since the effect on body weight gain was transient and did not impact the terminal body weight, these variations were not considered to be of biological importance.
There was no difference in body weight at the dose-level of 62.5 mg/kg/day.

There were no effects of treatment on female body weight or body weight gain at any dose-level during the premating period.

During gestation, the body weight gain of test item-treated animals was lower than in controls during days 7-14 (-15, -17 and -24%) and this
effect was still recorded during the last week of gestation in groups 3 and 4 (-12 and 18% vs. controls, respectively). Neither of these differences
reached statistical significance. In group 4 given 1000 mg/kg/day, the body weight difference recorded was considered to be due to one female
(U22974) who gained less weight secondary to a high pre implantation loss.

Food intake was unaffected by treatment.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There was no effect of treatment on mating at any dose-level. The apparent increase in the number of days taken to pair recorded in the high-dosegroup was not obvious since it was only 0.6 days higher than the control mean and was due to the contribution of only one female (U22973) whichrequired 13 days for mating because it was 11 days blocked in diestrus. Thus, a relationship to treatment with the test item was excluded.
The fertility index was unaffected by treatment.
All pregnant females had live pups.
No difficulties at delivery were observed in any female. The duration of gestation was similar in the control and the treated groups, and close to the normal value of 21 days.
The increase in mean pre-implantation loss in the high-dose group was due to one female (U22974) which had a low number of corpora lutea
(only 8), 62.5% of pre implantation loss and only delivered three pups.
There were no treatment-related effects on the post-implantation period, the increase in mean post implantation loss noted in the low-dose group being due to one female (U22951) which had 57.1% post implantation loss.
Taking into account the isolated case of the female No. U22974 which was considered to be fortuitous, it could be concluded that the repeated
administration of the test item had no influence on the delivery data recorded during this study.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination was performed in control and high-dose group animals.

Testes and epididymides
In both treated and control animals, the structures of tubules and interstitial cells were normal and did not show any
treatment-related changes.
Desquamation observed in tubules from occasional animals in control and treated groups were considered to be artefactual.
Focal atrophy/degeneration of tubules observed in one control and one treated animal was considered to be part of the normal background of
changes in the rat and without any relationship with treatment.
No treatment-related changes were observed in epididymides from treated animals when compared with controls.

Other organs: no treatment-related findings.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: NOAEL = highest tested dose, without relevant effects.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
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)

CLINICAL SIGNS (OFFSPRING)
Hematoma on head recorded in the pups was considered to be distributed among groups with no dose-related incidence, and as such, was notattributed to the test treatment.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test item was administered to male and female Sprague-Dawley rats by oral route (gavage), under the above detailed experimental conditions, at dose-levels of 62.5, 250 or 1000 mg/kg/day.

At 1000 mg/kg/day during the pre-mating period, there were no treatment-related clinical signs, and the body weight gain of males was transiently
lower than in controls in week 2 without consequences on the terminal body weight of males. There were no statistically significantly differences in
the body weight of test item-treated females when compared to control mean values. One out of ten females from the high dose group had a low
number of corpora lutea, high pre implantation loss and delivered only three pups, but this observation was not considered to be treatment-related.

There were no substance-induced effects on male or female reproductive performance or on the progeny of the parental rats at any dose-level.

Daily oral administration of the test item at the dose-level of 1000 mg/kg/day in the conditions of the study did not induce any treatment-related
changes in the examined testes and epididymides in males or in ovaries in females.

Based on the experimental conditions of this study and the isolated findings recorded at 1000 mg/kg/day, the dose-level of 1000 mg/kg/day was
considered to be the No Observed Adverse Effect Level (NOAEL) for parental toxicity and for reproductive performance (mating and fertility).
Executive summary:

The potential toxic effects of the test item were evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day post-partum (p.p.). This study should provide information on all aspecteds of reproduction and development.

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item by daily oral (gavage) administration for 15 days before mating, through mating, gestation and the beginning of the lactation period (until day 4 post-partum,

p.p.). The dose-levels were 62.5, 250 and 1000 mg/kg/day. Another group of 10 males and 10 females received the vehicle, purified water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg.

The concentration of test item in the dosage forms was checked at regular intervals.

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals throughout the study. Males were sacrificed after the end of the mating period (at least 5 weeks of treatment in total), females on day 5p.p..The adults were subjected to a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs. Designated organs from adult animals were weighed. In addition, the numbers ofcorpora luteaand implantation sites were recorded for each female. A microscopic examination of the reproductive organs (epididymides, ovaries with oviducts and testes) of the control and high-dose animals was performed. The pups were observed daily for clinical signs, sexed and weighed on days 1 and 5p.p..After their sacrifice on day 5p.p.,theywere examined to detect gross external abnormalities and a macroscopicpost-mortemexamination was performed.

The test item concentrations in the administered dosage forms were within the acceptable range of -9% to -4% of variation compared to the nominal values. There were no treatment-related clinical signs during the study,soft feces in 1/10 males given 250 or 1000 mg/kg/day and soiled urogenital area in 1/10 males given 1000 mg/kg/day being noted transiently during the pre-mating period. During the pre-mating period (days 1 to 15), males given 250 or 1000 mg/kg/day gained less weight than controls. Since these variations were transient and did not impacted the final body weight, there were not considered to be of toxicological importance. During the rest of the study, the body weight gain was unaffected and food consumption was similar between the test item‑treated male groups and the controls. During gestation, none of the variations of body weight gain recorded were statistically significantly different from the control values. Food consumption of test item-treated females was not affected during the study.

There were no effects of treatment on mating at any dose-level.The male and female fertility indices were unaffected by treatment;all pregnant females had live pups. The duration of gestation was similar in the control and test item-treated groups and close to the normal value of 21 days.

There were no effects of treatment on the mean number of live born pups or on pup death after birth. There were no gross external pup abnormalities in the control or test item-treated groups. The high pre-implantation losses recorded in a single female at 1000 mg/kg/day (which delivered only three pups) was considered to be isolated, and thus a relationship to treatment with the test item was unlikely. No differences of toxicological importance were noted in the male and female pup body weight gains. No relevant macroscopic findings were observed in the pups sacrificed on day 5 post‑partum. No treatment-related changes were observed in the testes or epididymides weights. There were no necropsy findings that could be ascribed to the test item treatment. At microscopic examination, there were no treatment-related changes in epididymides, testes or ovaries from treated animals when compared with controls.

The test item was administered to male and female Sprague-Dawley rats by oral route (gavage), under the above detailed experimental conditions, at dose-levels of 62.5, 250 or 1000 mg/kg/day. At 1000 mg/kg/day during the pre-mating period, there were no treatment-related clinical signs, and the body weight gain of males was transiently lower than in controls in week 2 without consequences on the terminal body weight of males.There were no statistically significantly differences in the body weight of test item-treated females when compared to control mean values.One out of ten females from the high‑dose group had a low number ofcorpora lutea, high pre‑implantation loss and delivered only three pups, but this observation was not considered to be treatment-related. There were no substance-induced effects on male or female reproductive performance or on the progeny of the parental rats at any dose-level. Daily oral administration of the test item at the dose-level of 1000 mg/kg/day in the conditions of the study did not induce any treatment-related changes in the examined testes and epididymides in males or in ovaries in females. Based on the experimental conditions of this study and the isolated findings recorded at 1000 mg/kg/day, the dose-level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental toxicity and for reproductive performance (mating and fertility).

 

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