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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:
From February 26,2010 to May 04,2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well described GLP compliant study conducted to recognised international test guidelines

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : 1, 2, 3, 6 Tetrahydrophthalic Anhydride (THPA)
- Molecular formula : C8H8O3
- Molecular weight : 152.1473
- Smiles notation : O=C1OC(=O)C2C1C/C=C\C2
- InChl : 1/C8H8O3/c9-7-5-3-1-2-4-6(5)8(10)11-7/h1-2,5-6H,3-4H2
- Physical state: White scales
- Lot/batch No.: SAT4209215
- Expiration date of the lot/batch: 03 June 2010
- Storage condition of test material: Room temperature in a desiccator

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Italy S.r.l.,
- Choice: The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities for reproductive toxicol ogy studies and there is ample experience and background data on this species and strain.
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 176 to 200 g for males and 151 to 175 g for females
- Housing:limited access rodent facility: in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor . Each cage tray held absorbent paper which was inspected and changed at least 3 times a week. During the mating period, animals were housed on the basis o f one male to one female in clear polycarbonate cages measuring 42x26x18 cm with a stainless steel mesh lid and floor . Each cage tray held abso rbent material which was inspected and changed daily.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42x26x18cm . Suitable nesting mater ial was provided and was changed at least 3 times a week.
- Diet: A commercially available laboratory rodent diet (4 RF 21) was offered ad libitum throughout the study.
- Water : Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period:19 days
- Health check: during the acclimatisation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22°C ± 2°C
- Humidity (%):55% ± 15%
- Air changes (per hr):15 to 25 air changes per hour
- Photoperiod :artificial light for 12 hours each day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The formulations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle :oral route was selected as it is a possible route of exposure of the test item in man.
- Amount of vehicle : The test item was mixed with corn oil to give the required concentrations of 10, 25 and 60 mg/ml.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until sperm identification and/or copulation plugs and/or indications of pregnancy were found.
The pairing combination of the animal which did not have positive identification of mating after 14 days of pairing was changed within its treatment group. The subsequent pairing was monitored for mating as described above until indications of pregnancy were evident
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in the range of 1 to 100 mg/mL. The proposed formulation procedure was checked in the range of 10 to 60 mg/mL to confirm concentration, homogeneity and stability and found to be within acceptance limits for concentration (90-110%) and homogeneity (CV <10%). The formulation was stable for at least 24 hours at room temperature. Samples of the formulations prepared on Day 1 and during Week 6 were analysed to check the homogeneity and concentration and found to be within the limits of acceptance (90-110% for concentration and CV <10% for homogeneity).
Duration of treatment / exposure:
Males:
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for a total of 6 consecutive weeks.
Females:
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods until Day 3 post partum.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/bw
Basis:
actual ingested
negative control
Remarks:
Doses / Concentrations:
100 mg/kg/bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg/bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg/bw
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on findings from similar studies with structural analogues of the test substance.
- Other:Control animals received the vehicle alone at the same dose volume.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Throughout the study, all animals were checked twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least once daily during the study. The interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
Males: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Females: on the day of allocation to treatment groups, weekly from the first day of treatment to mating, on Days 0, 7, 14 and 20 post coitum and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
At weekly intervals. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 0 post partum).

MATING:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until sperm identification and/or copulation plugs and/or indications of pregnancy were found. The pairing combination of the animal which did not have positive identification of mating after 14 days of pairing was changed within its treatment group. The subsequent pairing was monitored for mating as described above until indications of pregnancy were evident.

PARTURATION CHECK AND DURATION OF GESTATION:
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation periods were taken as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring). The day that offspring were first detected was considered as Day 0 post partum.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations:
testis weight, epididymis weight, sperm morphology of the seminiferous epithelium (staging of spermatogenic cycle)
Litter observations:
As soon as possible after parturition (Day 0 or 1 post partum), the total litter size (live and dead) was counted, sexed and examined for external abnormalities. Live pups were individually identified within the litter. All litters were weighed on Day 1 post partum. All litters were examined daily for dead and abnormal pups. The pups were also weighed on Day 4 post partum. All pups found dead were necropsied.
Postmortem examinations (parental animals):
All females including non-pregnant females were examined for:
a) external and internal abnormalities;
b) number of visible implantation sites (for pregnant animals);
c) number of corpora lutea (if detectable).
Uteri of non-pregnant females or with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Postmortem examinations (offspring):
All pups found dead in the cage were necropsied. All live pups were killed on Day 4 post partum and examined for:
a) external abnormalities;
b) sex confirmation by gonadal inspection.
Statistics:
For continuous variables the significance of the differences amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables were carried out by means of the Kruskal Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Statistical analysis of histopathological findings were carried out by means of the non-parametric Kolmogorov-Smirnov test.
Reproductive indices:
Males - Fertility Index (%)
Females - Fertility Index (%)
Males and females - Copulatory Interval; Copulatory Index (%)
Offspring viability indices:
Pre-birth loss; Pup loss at birth; Cumulative pup loss on Day 4; Sex ratio at birth and on Day 4 post partum

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:
no effects observed
Reproductive performance:
not examined

Details on results (P0)

CLINICAL SIGNS:
No significant clinical signs observed.

MORTALITY:
One female from the low-dose group (100 mg/kg/day) was humanely killed on Day 23 of gestation for difficulties in parturition.

MATING PERFORMANCE:
All females mated, even though mating was not detected in 1 mid-dose female.
The number of females with live pups on Day 4 post partum was 8 in each of the control and low dose groups and 9 in each of the mid- and high dose groups.

BODY WEIGHT:
Body weight and body weight gain of males were similar between groups. Very slight (less than 10%), sometimes statistically significant and/or dose-related reductions in body weight and body weight gain were observed in the treated females during the pre-mating treatment period. Slight but statistically significant reductions were still present in the high dose females during the gestation period.

FOOD CONSUMPTION:
No effects on food consumption were observed in the males. Slight and not dose-related reductions, in the range of 11% – 23%, were seen in the treated females during the pre-mating period. These reductions gradually recovered during the gestation period and were no longer present in the post partum period.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE:
No treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls.
The mean pre-coital interval and the number of copulation plugs were similar between control and treated groups.
A copulatory index of 100% was observed for both sexes from all groups.

ORGAN WEIGHTS:
Absolute and relative weight of testes, epididymides and ovaries were similar between groups.

NECROPSY:
The low-dose female killed for humane reasons on Day 23 of gestation showed red staining in the urogenital region with yellow mucoid contents in the amniotic sac. Pale or red colour was noted in the liver, kidneys and mesenteric lymph nodes.
No macroscopic observation was reported in the treated animals that could be considered treatment related.
Two control females, 1 low-dose, 1 mid-dose and 1 high-dose female were found not pregnant at necropsy. In addition, unilateral implantation was recorded in 1 low-dose female.

HISTOPATHOLOGY
The testes, epididymides and ovaries of control and high-dose animals were examined and on all abnormalities detected at post mortem examination.
No treatment-related changes were seen neither in males nor in females (testes, epididymides and ovaries) treated at 600 mg/kg/day.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. The minimal tubular cell degeneration, occasionally noted in control and treated males, did not alter the regularity in the spermatogenic cycle and was considered to be either incidental in origin or expression of spontaneous pathology.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
slightly increased in high-dose group
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)

Implantation, pre-birth loss data and gestation length
No differences in the number of implantation or pre-birth loss data were noted in treated animals compared to controls.
A prolonged gestation period, significantly different at statistical analysis from the control group, was noted in all treated groups, with a dose-related trend.
Litter data and sex ratio:
Although total litter size was slightly but significantly reduced at statistical analysis in animals dosed at 600 mg/kg/day, live litter size and pup loss were comparable between groups on Days 1 and 4 post partum. The statistically significant reduction in total litter size resulted in a statistically significant decrease in the number of females, although sex ratio (as % of males) was comparable between groups on Days 1 and 4 post partum.
Mean pup weight was slightly higher in the high dose animals than in controls on Days 1 and 4 post partum. However, no differences were observed in litter weight.

Pre-weaning clinical signs of pups:
No relevant findings that could be related to treatment were reported at clinical signs of pups during the lactation period.

Necropsy findings in pups:
No relevant differences between control and treated groups were noted at necropsy in the decedent pups.
No abnormalities were recorded in the pups sacrificed on Day 4 post partum

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
An effect on pregnancy, demonstrated by a prolonged gestation period and a smaller litter size, was the main treatment-related effect observed. These effects showed a dose related trend but were considered to have toxicological significance only at the highest dose level (600 mg/kg/day). A slight effect was also observed during gestation in the body weight of the high dose females. On the basis of the results obtained, the lower dose of 250 mg/kg/day was considered as the NOAEL (No Observed Adverse Effect Level).
Executive summary:

Reproductive and developmental toxicity has been investigated in a screening study conducted in accordance with OECD test methods. Groups of 10 males and 10 females received 1, 2, 3, 6 Tetrahydrophthalic Anhydride (THPA) at dosages of 100, 250 and 600 mg/kg/day. Males were treated for 2 weeks prior to pairing and during pairing of all females for a total of 6 consecutive weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partum.

 

An effect on pregnancy, demonstrated by a prolonged gestation period and a smaller litter size, was the main treatment-related effect observed. These effects showed a dose related trend but were considered to have toxicological significance only at the highest dose level (600 mg/kg/day). A slight effect was also observed during gestation in the body weight of the high dose females.

 

On the basis of the results obtained, the lower dose of 250 mg/kg/day was considered as the NOAEL (No Observed Adverse Effect Level).