Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening study according to OECD 421 with the registered substance, 4-MHHPA, showed no adverse effects on fertility. The NOAEL was determined to be 450 mg/kg bw/day.

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

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
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
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
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: 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.
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
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Well described GLP compliant study conducted to recognised international test guidelines.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The effects hexahydro-4 methylphthalic anhydride (4-MHHPA) 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 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 treatment.

On the basis of these findings, 450 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL). This study is acceptable and satisfies the guideline requirement for a reproduction and developmental toxicity screening study in rats (OECD 421).

Effects on developmental toxicity

Description of key information

Based on a prenatal developmental toxicity study in rats, conducted in accordance with OECD TG 414, the NOAEL is considered to be 460 mg/kg/day for development of offspring.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-24 to 2018-07-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment
Version / remarks:
24 July 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T404217186
- Manufacturing date: 05 Jul 2017
- Expiration date of the lot/batch: 04 Jul 2018

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.
Species:
rat
Strain:
Wistar
Remarks:
Hsd. Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Females: young adult and nulliparous females, ca. 9 weeks of age at start of the mating period; Males: experienced males, ca. 25-37 weeks of age at start of the mating period
- Weight at study initiation: The group averages of the body weight of the females were as similar as possible on the first day of gestation.
- Fasting period before study: none
- Housing: Before mating: 1-3 females per cage 1-2 males per cage; During mating: 1 male and 1-3 females / cage; During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage; in Type II polypropylene/polycarbonate cages, Bedding: Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Holzmühle 1, Germany
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water: tap water from municipal supply, as for human consumption from 500 mL bottle, ad libitum
- Acclimation period: 13 days for females, ca. 11 weeks for males

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 35 - 57 %
- Air changes: above 10 air exchanges/hour by central aircondition system
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil) in concentrations of 115 mg/mL, 60 mg/mL and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility from daily to every three days and stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil was used as vehicle, because the substance is hydrolytically unstable in water.
- Lot/batch no.: 1710-4753
- Concentration in vehicle: 20, 60 and 115 mg/mL
- Amount of vehicle: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study. Formulation samples prepared in sunflower oil were diluted with n-hexane and then analysed by a GC – FID method.
Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle and analyzed. The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 97 and 99 % of nominal concentrations at 1 and 280 mg/g in sunflower oil, respectively. Test item proved to be stable in the formulations for one day at room temperature and for three days in refrigerator (5 ± 3°C). A separate analytical report provided these results.
Details on mating procedure:
- Impregnation procedure: cohoused
- The males were paired to females in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieved at least six.
- M/F ratio per cage: 1 male and 1-3 females per cage
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
daily
Duration of test:
The animals were sacrificed on gestation day 20.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
Dose / conc.:
460 mg/kg bw/day (nominal)
No. of animals per sex per dose:
26 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on no findings (OECD 421) and limited toxic findings (OECD 407) obtained with 450 mg/kg bw/day in previous studies, and the dose range finding prenatal developmental toxicity study with the test material in the rat.
The 1000 mg/kg bw/day dose in the dose range finding study caused death in 2/7 females. Therefore the dose was reduced to 460 mg/kg bw/day. A further female died subsequently to dose reduction after having received two times 1000 mg/kg bw/day and one time 460 mg/kg bw/day. Furthermore, severe clinical symptoms, as reduced activity, hunched position, piloerection, low muscle tone and diarrhea, were observed in two females after the reduction to 460 mg/kg bw/day. In the OECD 407 study mild squamous hyperplasia of the forestomach mucosa was seen at 450 mg/kg bw/day. Based on these results, 460 mg/kg bw/day was selected as the maximum applicable dose for the OECD 414 main study neither causing maternal death nor severe toxicity. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily after dosing, when signs of toxicity were detected, animals were observed more frequently.
- Individual observation included the check of behavior and general condition.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the male animals was not measured. Body weights of sperm positive females were measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by decapitation after anesthetizing by an overdose Release® Pentobarbitat-sodium solution administered by an intraperitoneal injection.
- Organs examined: The abdomen was opened, the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted.

OTHER:
Examination for Sign of Implantation:
On gestation days 13 the sperm positive females were checked for the presence of vaginal bleeding which indicated the implantation of conceptuses. If negative on day 13, the examination was repeated on day 14 of gestation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Fetuses were removed from the opened uterus. Euthanasia of the fetuses was performed by hypothermia. The fetuses were sunk in a Petri-dish filled up with water. Spontaneous movement of fetuses was observed as a viability assessment. The fetuses were washed with tap water and randomly laid on a filter paper with written ordinal numbering. Bleeding from the umbilical cord after it is cut was observed also as a sign of viability. Each live fetus and its placenta was weighed individually (fetuses accuracy 0.01 g, placentas accuracy 0.001 g). The gender of the fetuses was determined according to the anogenital distance.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If significance was the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Indices:
Besides the data mentioned above the pre-implantation losses, post-implantation losses, sex distribution, the percentages of fetuses with external abnormalities, visceral abnormalities, and skeletal abnormalities were calculated.
Historical control data:
Historical control data of Hannover Wistar rats are available. The historical control study on teratology in Hannover Wistar rats was performed on 61 sperm positive females at the test facility in 2008 (Study no. 000.197.1437).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs observed in the females of all dose groups. Piloerection, reduced activity and hunched position were recorded for one dam on gestation day 18 and 19 in the control group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the females died in the course of the study
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight, body weight gain in the different periods, corrected body weight of the females was similar in all groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Minimal but statistically significant decreases of the food consumption were observed in the test item treated groups if compared to the control. This was indicated before the treatment period (from gestation day 3 to 5) and in the treatment period (from gestation day 11 to 17) in the 80 mg/kg bw/day dose group and from gestation day 5 to 11 in all groups. There was no dose response seen and these differences were considered as biologically not relevant and unrelated to the treatment.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related necropsy findings were observed.
Reddish mottled lungs were recorded for 6, 9, 11 and 4 does respectively in the control, 80, 240 and 460 mg/kg bw/day group without a dose response. Pinhead sized haemorrhages were recorded in the lungs in single cases (one dam each in the test item treated groups). Both observations were not attributed to the test item. There were no macroscopic alterations recorded for the dams during necropsy in the experimental groups. Blood was observed in the uterus for one control dam.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no increase indicated in the mean percent of pre- and postimplantation loss (early embryonic, late- and fetal death). Moreover the CH square test indicated a statistically significant reduction (p<0.05) in the postimplantation loss in the low and high dose group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
The mean number of implantations, the sex distribution of the fetuses as well as in the mean number of viable fetuses was similar in all dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
460 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related differences in the body weight of the fetuses and placental weight.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable fetuses was similar in all dose groups.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no malformations found in the test item treated groups. Two fetuses in the control group had retrognathia, neck edema or whole body edema and mal-rotated forelimb/s.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of the affected litters was 1 in each experimental group.
A split sternebral cartilage in the 240 mg/kg bw/day group, a bipartite cartilage of the thoracic centrum, a hemicentric and cartilageously dumb-bell shaped lumbar centrum both in the low dose group and one multiply malformed fetus in the control and high dose group were found. The control fetus had a short mandible, a misshapen (wider) and split sternum, abnormal articulation of sternoclavicular joint, misshapen clavicle and scapula, bipartite cartilage of cervical VII vertebral centrum, hemicentric ossification of thoracic V vertebral centrum, a dumb-bell shaped or/and bipartite cartilage of thoracic IX and XII centrum respectively. The fetus in the high dose had a misshapen xiphoid cartilage with a hole, bipartite cartilage and hemi-centric ossification of thoracic III centrum as well as a bent left scapula and bentness of both humeri. The malformations found occurred sporadically with low incidence or/and without a dose response. Furthermore most of these changes occurred in control fetuses without a relationship to the treatment according to the historical control data. Therefore these malformations were judged to be unrelated to an effect of the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of the affected litters was 1 each in the test item treated groups.
Microphthalmia was found in one fetus in the mid dose and one in the high dose. Situs inversus totalis was observed in one fetus in the low dose group. Microphthalmia as well as situs inversus totalis occur sporadically with low incidence in control fetuses according to the experience of this laboratory which is in line with the historical control data. Considering also the low incidence, these malformations were judged as incidental, non-treatment related.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Placentas, amnion:
Edema and paleness, both for two fetuses in the control group and latter for one in the high dose group was recorded.
Visceral variations:
Hydroureter, observed in control and 240 mg/kg bw/day groups, without a dose response, and slightly rounded apex cordis in one fetus in the 460 mg/kg bw/day were evaluated as variations. Considering the low incidences and/or the lack of dose response these variations were not attributed to the treatment.
Skeletal variations:
There was a statistical significant increase (p<0.05) in the occurrence of litters with bipartite or/and asymmetric thoracic centrum (evaluated as variations during the skeletal examination) at 240 mg/kg bw/day but no dose response was indicated.
Details on embryotoxic / teratogenic effects:
There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations and variations found.
Key result
Dose descriptor:
NOAEL
Effect level:
460 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: PREGNANCY DATA OF FEMALES, MORTALITY, MALFORMATIONS

Dose groups

Control

80 mg/kg bw/day

240 mg/kg bw/day

460 mg/kg bw/day

Number of sperm positive females

26

26

26

26

Number of females with no implantation and no corpora lutea

3

6

4

6

Number of females with no implantation but corpora lutea

2

1

1

2

Number and percent of females with implantation (pregnants)

21

80%

19

73%

21

80%

18

69%

Number of females with total post-implantation loss

0

0

0

0

Number of pregnant females died due to toxicity

0

0

0

0

Number of pregnant females excluded due to a technical error (determination of mating day)

1

0

0

1

Number of females with live fetuses (number of litters)

20

19

21

17

Number and percent of litters with malformed fetuses

2

10%

2

10.5%

2

9.5%

2

11.8%

 

Table 2: SUMMARY OF CLINICAL SIGNS AND NECROPSY FINDINGS OF DAMS

 

control

80 mg/kg bw/d

240 mg/kg bw/d

460 mg/kg bw/d

No of animals

20

19

21

17

Clinical Symptoms

 

none

19

19

21

17

piloerection

1 *

0

0

0

Reduced activity

1 *

0

0

0

Hunched posture

1 *

0

0

0

Necropsy findings

 

No macroscopic alterations

13

9

9

12

Reddish mottled lungs

6

9

11

4

Pinhead-sized haemorrhages in the lungs

0

1

1

10

blood in uterus

1 *

0

0

0

* the same animal

 

Table 3: SUMMARY OF GRAVID UTERINE WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF DAMS

 

Control

80 mg/kg bw/day

240 mg/kg bw/day

460 mg/kg bw/day

Gravid uterine weight(g)

MEAN

62.5

65.4

62.5

67.9

 

SD

12.06

7.90

14.48

10.60

 

n

20

19

21

17 NS

Corrected body weight(g)

MEAN

262.0

258.5

259.2

255.5

 

SD

26.92

16.83

12.43

16.53

 

n

20

19

21

17 NS

Corrected

MEAN

50.0

47.5

46.1

44.8

body weight gain(g)

SD

26.37

11.76

9.49

12.84

 

n

20

19

21

17 NS

NS = Not Significant

 

 

Table 4: PREGNANCY DATA OF FEMALES, MORTALITY, MALFORMATIONS

Dose groups

Control

80 mg/kg bw/day

240 mg/kg bw/day

460 mg/kg bw/day

Number of sperm positive females

26

26

26

26

Number of females with no implantation and no corpora lutea

3

6

4

6

Number of females with no implantation but corpora lutea

2

1

1

2

Number and percent of females with implantation (pregnants)

21

80%

19

73%

21

80%

18

69%

Number of females with total post-implantation loss

0

0

0

0

Number of pregnant females died due to toxicity

0

0

0

0

Number of pregnant females excluded due to a technical error (determination of mating day)

1

0

0

1

Number of females with live fetuses (number of litters)

20

19

21

17

Number and percent of litters with malformed fetuses

2

10%

2

10.5%

2

9.5%

2

11.8%

 

Table 5: RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS

(percentile litter means and SD)

Dose groups

Control

80 mg/kg bw/day

240 mg/kg bw/day

460 mg/kg bw/day

EXTERNAL EXAMINATION

Litters examined

N

20

19

21

17

Fetuses examined

N

215

217

226

205

Fetuses

with abnormalities

Mean

6.2

1.8

8.5

5.8

SD

22.41

3.59

22.40

11.90

Variation

Mean

5.4

1.8

8.5

5.8

SD

22.34

3.59

22.40

11.90

Malformation

Mean

0.8

0.0

0.0

0.0

SD

3.44

0.00

0.00

0.00

Retarded in body weight

Mean

5.8

1.8

8.5

5.8

SD

22.44

3.59

22.40

11.90

VISCERAL EXAMINATION

Litters examined

N

20

19

21

17

Fetuses examined

N

109

109

115

104

Fetuses

with abnormalities

Mean

0.8

0.9

3.5

1.7

SD

3.73

3.82

9.54

6.93

Variation

Mean

0.8

0.0

2.9

0.8

SD

3.73

0.00

9.34

3.46

Malformation

Mean

0.0

0.9

0.6

0.8

SD

0.00

3.82

2.73

3.46

SKELETAL EXAMINATION

Litters examined

N

20

19

21

17

Fetuses examined

N

106

108

111

101

Fetuses

with abnormalities

Mean

16.8

9.5

16.7

11.4

SD

26.02

15.02

23.38

15.80

Variation

Mean

16.0

7.8

15.6

10.2

 

SD

24.56

11.58

23.58

14.19

Malformation

Mean

0.8

1.8

1.2

1.2

SD

3.73

7.65

5.46

4.85

NS

No significant findings.

Remarks:

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control DN = Duncan's multiple range test

 

Table 6: RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS

(sum, %)

 

 

Control

80 mg/kg bw/day

240 mg/kg bw/day

460 mg/kg bw/day

EXTERNAL EXAMINATION

Litters examined

N

20

19

21

17

Fetuses examined

N

215

217

226

205

Fetuses

with abnormalities

N

7

4

12

12

%

3

2

5

6

Variation

N

5

4

12

12

%

2

2

5

6

Litters

N

2

4

7

4

%

10

21

33

24

Malformation

N

2

0

0

0

%

1

0

0

0

Litters

N

1

0

0

0

%

5

0

0

0

Retarded in body weight

N

6

4

12

12

%

3

2

5

6

Litters

N

2

4

7

4

%

10

21

33

24

VISCERAL EXAMINATION

Litters examined

N

20

19

21

17

Fetuses examined

N

109

109

115

104

Fetuses

with abnormalities

N

1

1

5

2

%

1

1

4

2

Litters

N

1

1

3

1

%

5

5

14

6

Variation

N

1

0

4

1

%

1

0

3

1

Litters

N

1

0

2

1

 

%

5

0

10

6

Malformation

N

0

1

1

1

%

0

1

1

1

Litters

N

0

1

1

1

%

0

5

5

6

SKELETAL EXAMINATION

Litters examined

N

20

19

21

17

Fetuses examined

N

106

108

111

101

Fetuses

with abnormalities

N

15

10

14

12

%

14

9

13

12

Litters

N

10

7

12

7

%

50

37

57

41

Variation

N

14

8

13

11

%

13

7

12

11

Litters

N

10

7

11

7

%

50

37

52

41

Malformation

N

1

2

1

1

%

1

2

1

1

Litters

N

1

1

1

1

%

5

5

5

6

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

 

Conclusions:
Oral treatment of pregnant Hsd. Han: Wistar rats from gestation day 5 up to day 19 with the test item at the dose levels of 80, 240 and 460 mg/kg bw/day did not cause maternal toxicity or signs of developmental toxicity. The No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity (including teratogenicity) was 460 mg/kg bw/day.
Executive summary:

The test item was examined for its possible prenatal developmental toxicity in a GLP compliant study according to OECD 414. Groups of 26 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 80, 240 and 460 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. Concentrations of the test item in the dosing formulations varied in the acceptable range between 99 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. The body weights and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.In total, on gestation day 20 there were 20, 19, 21 and 17 evaluated litters in the control, 80, 240 and 460 mg/kg bw/day group respectively.None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in all dose groups. No treatment related necropsy findings were observed. There were no treatment related changes in food consumption or body weights indicated. The number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations found. Based on these observations the No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity (including teratogenicity) was 460 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
460 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Well described GLP compliant study conducted to recognised international test guidelines.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Hexahydro-4 methylphthalic anhydride (4 -MHHPA) was examined for its possible prenatal developmental toxicity in a GLP compliant study according to OECD 414. Groups of 26 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 80, 240 and 460 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle sunflower oil. During the study, mortality was checked and clinical observations were performed. The body weights and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.In total, on gestation day 20 there were 20, 19, 21 and 17 evaluated litters in the control, 80, 240 and 460 mg/kg bw/day group respectively.

None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in all dose groups. No treatment related necropsy findings were observed. There were no treatment related changes in food consumption or body weights indicated. The number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations found.

Based on these observations the No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity (including teratogenicity) was 460 mg/kg bw/day.

Supporting studies:

A dose range finding screen prior to the conduct of the full prenatal developmental toxicity study (OECD 414) was conducted. Groups of seven spermium-positive female Hsd. Han: Wistar rats were

treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000/460 mg/kg bw/day (the dose level of the high dose was reduced after mortality was observed) respectively from day 5 up to and including day 19 post coitum. A control group of seven sperm positive females was included and the animals were given the vehicle sunflower oil. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm were detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally.

The highest dose level of 1000 mg/kg bw/day of the test item caused clinical signs and death of two dams after treatment from gestation day 5 to 7. The test item caused death in a further dam after two treatments with 1000 mg/kg bw/day and one treatment with the reduced 460 mg/kg bw/day dose. Death of a single dam in the 300 mg/kg bw/day dose group was caused by misgavage and therefore is not test item related. During treatment from gestation day 5 to 19 with the dose level of 1000/460 mg/kg bw/day the test item caused clinical signs in one dam and in one female with total implantation loss even after reduction of the dose level. Similar clinical signs and necropsy findings were observed in one animal in the 300 mg/kg bw/day dose group. A test item related effect at 300 mg/kg bw/day was not considered due to the low incidence. There was no reduction in the food consumption and body weight parameters attributed to the test item considering the lack of dose response. The increase of early embryonic death in the 1000/460 mg/kg bw/day dose group was caused by one female with total post-implantation loss. The effect was considered treatment related, most likely secondary to the severe maternal toxicity demonstrated by the strong clinical symptoms. The mean number of implantations, viable fetuses per litters, percentage preimplantation

loss, late resorptions and fetal death were not affected by the test item. The treatment of the maternal animals did not result in reduced body weight of fetuses or increase of variations including

body weight retardation. The test item caused no malformations in the fetuses.

Based on the maternal toxicity and effects secondary to maternal toxicity this dose range finding study determined doses of 80, 240 and 460 mg/kg bw/day to be suitable for the main study (OECD 414).

Effects on fertility, pregnancy and early lactation of the offspring have also 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 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 treatment. On the basis of these findings, 450 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL).

Conclusion:

No adverse effects regarding development of offspring have been identified and the NOAEL for both maternal and developmental toxicity was determined to be 460 mg/kg bw/day for hexahydro-4 methylphthalic anhydride (4-MHHPA).

Justification for classification or non-classification

The results of reproductive and developmental toxicity testing show that 4-MHHPA classification and labelled according to Regulation (EC) No 1272/2008 (CLP) is not required.