Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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 December 22,2009 to March 07,2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T408209159
- Manufacturing date: 08 Jun 2009
- Expiration date of the lot/batch: 08 Jun 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed. Keep in a dry, cool and well ventilated place.
- Storage at testing facility: 5 years

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation:6 to 7 weeks old.
- Weight at study initiation: 187 to 208 g for males and 160 to 177 g for females
- Housing: limited access rodent facility. It is a clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor .Each c age tray held absorbent paper which was inspected and 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 : 12 hour per day by artificial light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The formulations were prepared daily. Formulation samples were constantly stirred from preparation until administration.

VEHICLE

- Concentration in vehicle:The test item was administered orally by gavage at a dose volume of 10 ml/kg body weight. Control animals received the vehicle alone at the same dose volume.
- Amount of vehicle : The required amount of test item was suspended in corn oil to give the required concentrations of 5, 15 and 45 mg/ml.

Details on mating procedure:
- M/F ratio per cage: 1/ 1
- Length of cohabitation: Until successful mating (up to14 days)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear; referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged : Females transferred to individual clear polycarbonate solid bottomed cages measuring 42x26x18 cm.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was verified in the range of 1 to 200 mg/mL to be at least 24 hours at room temperature.
The formulation procedure was checked in the range of 5 to 45 mg/ml by analysis for concentration and homogeneity.
Samples of the formulations prepared on Day 1 and at the end of treatment were analysed to check for homogeneity and concentration. Results of the analyses were within the limits of acceptance for suspensions (90-110% for concentration and CV <10% for homogeneity). .
Duration of treatment / exposure:
Each group comprised 10 male and 10 female rats. The group identification and animal numbers assigned to the treatment are summarised below:

Group Treatment Level
Number (mg/kg/day)+

1 0 Control
2 50 Low
3 150 Medium
4 450 High
Frequency of treatment:
Males were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy achieving a total of 42 days of treatment.
Females were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to and including Day 3 post partum.
Details on study schedule:
- Age at mating of the mated animals in the study:6/7 weeks old
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
The test item was administered orally by gavage at a dose volume of 10 ml/kg body weight.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Mortality:
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Clinical signs :
All clinical signs were recorded for individual animals, once before commencement of treatment and once daily during the study, with the exception of days 14 and 15 of the pre mating phase in which an additional observation was recorded in the high dose group of animals. Each animal was observed and any clinical signs were recorded. Observations were performed taking into consideration the presence of post-dose reactions.
Body weight :
Males:
Animals were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Females:
Animals were weighed 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.
Body weight of females performed at weekly interval during the pairing period was not reported, but the data will be archived with all other raw data.
Food consumption:
Food consumption was recorded at weekly intervals, by each cage of rats from allocation to pairing. 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). Erroneously was not recorded the food consumption of female no. 23 was, erroneously, not recorded on Day 4 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. The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis weight, epididymis weight, morphology of seminiferous epithelium (staging of spermatic 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):
The clinical history of the animal was studied and a detailed post mortem examination was conducted for the following:
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 impl
antation

Organ weights:
From all animals completing the scheduled test period the epididymides, testes and ovaries were weighed.

Tissues fixed and preserved
Samples of following tissues were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Bouin's solution and all preserved in 70% ethyl alcohol): abnormalities, coagulating glands, epididymides, ovaries prostate gland, seminal vesicles, testes, uterus/cervix and vagina.

Histopathological examination:
Any abnormalities observed at necropsy, the epididymides, testes and ovaries were examined. The testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS) for morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle).
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 the following:
a) external abnormalities;
b) sex confirmation by gonadal inspection
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was 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 was carried out by means of the non-parametric Kolmogorov-Smirnov test.
The mean values and standard deviations were calculated from actual values in the computer without rounding off.
Reproductive indices:
Males
Fertility Index (%) = [(no. of males which induced pregnancy )/(no. of males paired)] x 100

Females
Fertility Index (%) = [(no. of pregnant females)/(no. of females paired)] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
In the pre-pairing period no signs of toxicological relevance were detected in either sexes. Salivation was the most relevant clinical sign detected in mid- and high dose animals of both sexes, after 2 weeks of treatment during the pairing period. This sign was also noted in 9 out of 10 high dose females during the gestation period. No relevant signs were recorded during the post partum period.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were similar between groups in both sexes during the whole treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Slight decrease in food consumption was detected in treated and control animals at the end of the pre-pairing period.
No changes were noted in females during post coitum and post partum periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were seen in the males or in the females (testes, epididymides and ovaries) dosed at 450 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.
Lesions reported in control and treated animals as severe pyelonephritis and hydronephrosis in the kidneys with consequent distension and epithelial hyperplasia of the ureter associated with chronic inflammatory reaction extended to the cervix mass (high dose female), testicular tubular cell degeneration or hydrometra in the uterus were considered to be either incidental in origin or expression of spontaneous pathology, often seen in this species under experimental conditions.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls.
The pairing combination of individual females from the control, low dose and mid-dose groups which did not show positive identification of mating after 14 days of cohabitation with the first selected male, was changed and another male, within each treatment group, was selected for a second pairing.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
All control and treated females mated, although mating with the first male was not detected for 2 females which gave birth during the second pairing period.
One mid-dose male did not mate after the maximum period allowed (14 days). In addition, 2 control males and 2 high dose males did not induce pregnancy. No relevant differences were observed in treated males and females reproductive parameters compared to controls.
A slight increase in pre-coital interval was noted in treated groups compared to controls. The difference did not show a clear dose-relationship and was considered not to be of toxicological relevance.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Pre-weaning clinical signs of pups: No relevant findings that could be considered treatment-related were reported in pups during the lactation period.The increased number of decedent pups noted in the mid-dose group was considered to be
incidental.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in pups: No milk in the stomach and autolysed abdominal organs were generally observed in pups which died during the lactation period. No abnormalities were detected in the pups sacrificed on Day 4 post partum
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects observed.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
No significant changes and no treatment-related effects were detected in males and females during the in vivo phase.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.
Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.
Findings at macroscopic and microscopic examinations, in both males and females, did not reveal differences between groups that could be related to the administration of the test item.
On the basis of the results obtained, the dosage of 450 mg/kg/day is considered as the NOAEL (No Observed Adverse Effect Level) for this study.
Executive summary:

The effects hexahydro-4 methylphthalic anhydride on fertility, pregnancy and early lactation of the offspring have been investigated in a reproduction/developmental toxicity screening study conducted according to OECD test methods. Groups of rats were dosed by oral gavage at levels of 50, 150 and 450 mg/kg/day. Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods and for 4 days post partum. 

No significant changes and no treatment-related effects were detected in males and females during thein vivophase. Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls. Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.

Findings at macroscopic and microscopic examinations, in both males and females, did not reveal differences between groups that could be related to treatment.

On the basis of these findings, 450 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL).