Tetrahydromethylphthalic anhydride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-06 to 2021-05-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydro-4-methylphthalic anhydride

EC Number:

243-072-0

EC Name:

Hexahydro-4-methylphthalic anhydride

Cas Number:

19438-60-9

Molecular formula:

C9H12O3

IUPAC Name:

5-methylhexahydro-2-benzofuran-1,3-dione

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: females: ca. 9 weeks of age at start of the mating period; males: 28-29 weeks of age at start of the mating period
- Weight at study initiation: females: 177-249 g
- Fasting period before study: no
- Housing: before mating: 1-2 females/cage, 2 males/ cage; during mating: 1 male and 1-2 females/cage; during gestation: 2 sperm positive females/ cage, if not possible 1 sperm positive female/ cage; cages: Type II polypropylene/polycarbonate; bedding: Safe 3/4-S Fasern Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany)
- Diet: ad libitum, ssniff SM R/M " breeding and maintenance autoclavable complete feed for rats and mice " produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: females: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 24.5
- Humidity (%): 37 - 62
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

Sunflower oil (Helianthi annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 115 mg/mL, 153.75 mg/mL and 200 mg/mL in the laboratory of Toxi-Coop Zrt daily to every three days.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil was used as vehicle, because the substance is hydrolytically unstable in aqueous media. Sunflower oil proved to be suitable for treatment.
- Concentration in vehicle: 115, 153.75, 200 mg/mL
- Amount of vehicle: A constant treatment volume of 4 mL dose preparation/kg body weight was administered in all groups.
- Lot/batch no.: 8006332001

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 the Test Facility two times during the study by a GC-MS method. Five samples from each concentration were taken from different places of the test item formulations for analysis of concentration and homogeneity. Similarly, five samples were taken from the vehicle (Control) and analyzed.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male/ one to two females
- Length of cohabitation: 2-4 h
- After unsuccessful pairing replacement of first male by another male with proven fertility: not performed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

14 days
The sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

daily

Duration of test:

The animals were sacrificed by anaesthesia on gestation day 20.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

800 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

615 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

460 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

A total of 104 males and 130 females for mating were used to achieve the sufficient number of females per group of at least 22 spermium positive females per group or at least 16 litters per group (28 to 30 spermium positive females and 19-23 litters per group achieved).

The number of mated and pregnant females was as follows:
Control: mated: 28, pregnant: 23
460 mg/kg bw/d: mated: 28, pregnant: 21
615 mg/kg bw/d: mated: 30, pregnant: 20
800 mg/kg bw/d: mated: 28, pregnant: 23

NB: Total pre-implantation losses were accounted as non-pregnant.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finding study, where oral treatment of pregnant Han: WIST rats with the dose level of 900 mg/kg bw/day of the test item caused death of three dams and clinical signs of the animals, as well as moderate reduction of food consumption and lower body weight gain. The increase of fetal death in the 900 mg/kg bw/day dose group due to one female with total post-implantation loss and moderately lower mean number of viable fetuses was considered treatment related, most likely secondary to the severe maternal toxicity. Less significantly lower food consumption and body weight gain including corrected body weight gain at 700 mg/kg bw/day were not unambiguously attributed to an effect of the test item. Slightly lower body weight of fetuses at 900 and 700 mg/kg bw/day (latter only females) was not proved to be due to the treatment with the test item. The test item caused neither malformations, nor increase of fetal variations or placental abnormalities.
A dose level of 800 mg/kg bw/day was selected as highest dose level in this main study with the aim to induce some maternal/developmental toxicity but not death or severe suffering. The low dose of 460 mg/kg bw/day was chosen to induce no toxic effect. The mid dose of 615 mg/kg bw/day was interpolated geometrically.
- Rationale for animal assignment (random): The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. If possible, females paired with the same male were allocated to different groups on the same mating day.
- Fasting period before blood sampling for dam thyroid hormones: No fasting period described.
- Time of day for dam blood sampling: in the morning on the day of necropsy

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day from g.d. 0 to 20, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were observed more frequently.
Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.
Observations for signs of morbidity and mortality were made twice daily from g.d. 5 to 19, at the beginning and end of the working period, and once on g.d. 20.

DETAILED CLINICAL OBSERVATIONS: Yes, see above.

BODY WEIGHT: Yes for females
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

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
- Organs examined: The uterus with cervix and the left ovary 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.
Thyroid glands, together with the parathyroid glands were removed and fixed in 4 % (v/v) buffered formalin solution for histopathological evaluation. They were weighed and recorded with a precision of 0.001 g after fixation.
Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % (v/v) buffered formalin solution at necropsy for possible histological examination. Control organs were fixed in 4 % (v/v) buffered formalin solution and preserved for comparison.

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
- Other: Live fetuses and fetal death were counted.

Blood sampling:

Blood samples collected for TSH and Thyroid Hormones (FT3 and FT4) measurements were drawn from all sperm positive females from the sublingual vein in the morning on the day of necropsy.
- Plasma: No
- Serum: Yes
- Volume collected: at least 1.0 mL blood

Fetal examinations:

- 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
- Anogenital distance of each fetus was determined.

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
Means and standard deviations and/or percentages were calculated.
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 the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Indices:

- Pre-implantation loss: ((Number of corpora lutea - Number of implantations)/Number of corpora lutea)*100
- Post-implantation loss: ((Number of implantations - Number of live fetuses)/Number of implantations)*100
- Sex distribution: (Number of Male (Female) fetuses/Number of fetuses)*100
- External abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses)*100
- Visceral abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses examined)*100
- Skeletal abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses examined)*100

Historical control data:

Historical control data on prenatal developmental toxicity from studies in the rat strain conducted in the same laboratory from 2017-2021 were included in the study report and used as reference.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Digging in the bedding after treatment, salivation, hunched back, piloerection, reduced activity and noisy breath were observed in the 615 and 800 mg/kg bw/day groups with moderate incidences. Hunched back and noisy breathing were observed also in one animal in the 460 mg/kg bw/day group. Dyspnoea was recorded for one female in the 615 mg/kg bw/day group. The clinical signs described for the 615 and 800 mg/kg bw/day groups were considered adverse and attributed to the treatment with the test item. The single finding in one animal of the low dose group is considered incidental.
In addition, one control female had swelling in the armpit without a toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female died in the course of the study in the 800 mg/kg bw/day group. It was found dead in the morning on g.d. 12. This female had clinical signs such as hunched back and piloerection from g.d. 10 onwards as well as reduced activity on g.d. 11. Digging in the bedding was also observed from g.d. 9 to 11.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was statistically significantly (p<0.01) lower in the 800 mg/kg bw/day group between g.d. 8 and 11, as well as g.d. 5 and 20 (p<0.05). Also, the corrected body weight gain was statistically significantly lower in this group (p<0.05). Statistics revealed also significance (p<0.05) in the 615 mg/kg bw/day group between g.d. 5 and 20. The statistically more pronounced lower body weight gain in the 800 mg/kg bw/day group between g.d. 8 and 11 was largely related to the female found dead on g.d.12. Overall, the lower body weight gain in the 800 and 615 mg/kg bw/day groups was attributed to an effect of the test item.
No differences in the 460 mg/kg bw/ day group were detected. Furthermore, no significant differences either in the body weight (including corrected body weight) of the dams of all treatment groups were observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower food consumption was observed in the 800, 615 and 460 mg/kg bw/day group in a dose-dependent manner between g. d. 8 and 11 (p<0.01 (-10 and -22 %) in the 460 and 800 mg/kg bw/day groups respectively and p<0.05 (-14 %) in the 615 mg/kg bw/day group). The reduction in the 800 mg/kg bw/day group between g.d. 8 and 11 was mostly caused by the female found dead on g.d.12. Between g.d. 11 and 14 statistical significance was indicated only in the 460 (-8 %) and 615 (-12 %) mg/kg bw/day groups and not in the 800 mg/kg bw/day group. Between g.d. 14 and 17 the values were statistically significantly lower only the 615 mg/kg bw/day group (p<0.05, -8 %) without a dose response.
The reduction in the 460 mg/kg bw/day dose group could be largely related to the low individual data of female No.: 181 between g.d. 5 and 11 which also exhibited clinical signs as hunched back on g.d. 10.
No effects on food consumption were observed at the other gestation days or groups.
If the percentages were taken into account, the reduced food consumption in the 800 mg/kg bw/day group between g.d. 8 and 11 was considered as a significant test-item related effect (-22%).
The lower food consumptions between g.d. 8 and 14 in the 615 mg/kg bw/day group and between g.d. 11 and 14 in the 800 mg/kg bw/day group were judged as slight or moderate effects of the test item (between 11 and 14 %).
The slight differences in the 460 mg/kg bw/day group between g.d. 8 and 14 were considered as not adverse, considering that the percentages were between 8 and 10 %.

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

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower values were measured in the 800 (p<0.05, -13 %), 615 (p<0.01, -15 %) and 460 (p<0.01, -16 %) mg/kg bw/day groups for the free thyroid hormone level FT4. No dose-dependence was observed and all values were within the range of the historical control data (mean of 0.0129 and 22%CV). Therefore, these differences were considered to be indicative of biological variation and have no toxicological relevance. The FT3 values were also statistically significantly lower in the 615 and 460 mg/kg bw/day groups (p<0.01 both), but not in the 800 mg/kg bw/day group, hence without a dose response. The TSH hormone levels were similar in all groups. As no dose response relationship was observed for FT3 and TSH levels, the described effects are considered to be not test item related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean thyroid weight values were similar in the experimental groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lung emphysema, congestive liver and bloody jelly content in the ileum was recorded for one animal treated with 800 mg/kg bw/day which died before scheduled necropsy on gestation day 12.
Other findings such as pinhead-sized haemorrhages in the lungs, dilated renal pelvis occurred with low incidences and without a dose response. Therefore, they were considered as unrelated to the test item. In the control group a liver that was fused with the diaphragma or a subcutan formation was observed in one dam each.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item- related lesions observed upon histological examinations of the thyroid tissue.
The histological examination of the animal which died before scheduled necropsy in the 800 mg/kg bw/day group revealed multifocal myocardial fibroblast proliferation accompanied with atrophy of affected myocytes in the heart, alveolar emphysema in the lungs, and congestion in the liver as well as in the small intestines as cause of death. Lung emphysema, congestive liver and bloody jelly content in the ileum was recorded for one animal treated with 800 mg/kg bw/day which died before scheduled necropsy on gestation day 12. Findings such as pinhead-sized haemorrhages in the lungs, dilated renal pelvis occurred with low incidences and without a dose response. Therefore, they were considered as unrelated to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

One female in the 800 and one in the 615 mg/kg bw/day group had total postimplantation loss. In the 615 mg/kg bw/day group two females and in the 800 mg/kg bw/day group as well as in the control one female had total preimplantation loss.
If the females with total pre- and post-implantation loss were included to the data evaluation, the post-implantation loss was statistically significantly higher in the 800 mg/kg bw/day group (for mean value: p<0.05 by U-test and for sum p<0.01 by Chi square).
According to the Chi square test also early embryonic death (sum) increased statistically significantly (p<0.01) for those animals. Preimplantation loss and intrauterine mortality (summarized pre- and post-implantation loss) were statistically significantly higher (p<0.01) for sum value in the 615 mg/kg bw/day group. No dose dependency was observed.
Statistically significantly lower sum values were seen in the 460 mg/kg bw/day group (pre-implantation loss and total intrauterine mortality; p<0.05 by Chi square) without a toxicological relevance. There were no statistically significant differences in the mean number of implantations and late embryonic death.
If the females with total pre- and post-implantation loss were excluded, difference from the control values decreased and there was no statistical significance indicated in any parameters.
For the slight increase of post-implantation loss (early and late resorptions) including the occurrence of total post-implantation loss in one female both in the 800 and 615 mg/kg bw/day group a treatment relationship cannot be excluded.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "Pre- and post-implantation loss".

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Please refer to "Pre- and post-implantation loss".

Dead fetuses:

no effects observed

Description (incidence and severity):

No fetal death was observed in all groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number and percentage of pregnant females compared to mated females showed similar values in all groups.
Control: mated: 28, pregnant: 23/ 82.1%
460 mg/kg bw/d: mated: 28, pregnant: 21/ 75.0 %
615 mg/kg bw/d: mated: 30, pregnant: 20/ 66.7 %
800 mg/kg bw/d: mated: 28, pregnant: 23/ 82.1 %

Other effects:

no effects observed

Description (incidence and severity):

The values for placental weight were similar in the treatment groups and the control group.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

pre and post implantation loss

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the body weight of the fetuses. The values for body weight were similar in the treatment groups and the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses was equal among the groups.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The values for absolute and normalized anogenital distance were similar in the treatment groups and the control group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of evaluated fetuses was 266, 243, 209 and 229 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively.

Variations
There was no variation observed.

Malformations
There were no malformations found.

Growth retardation
The incidence of body weight retarded fetuses was higher in the 800 and 615 mg/kg bw/day groups (at a similar level), however statistics revealed no significance. Hence, a test item effect was not proved.

Placentas
Larger placentas and or with wider margin were found in one litter in the 800 mg/kg bw/day group. A test item relationship remains unclear as this observation was limited to only one single dam. It should be noted, that two fetuses to which these placentas belonged were malformed (bent scapula or and slightly thicker humerus).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 129, 122, 104 and 114 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. The incidence of fetuses with abnormalities and variations was statistically significantly higher (p<0.05 and p<0.01 respectively) in the 615 mg/kg bw/day group according to the Chi square test. No statistical difference was observed if the number of litters affected was considered. The mean percent per litter for total variations was also significantly higher (p<0.05) according to Duncan's multiple range test in the 615 mg/kg bw/day group. There was no dose response indicated in variations, abnormalities and in the incidence of malformations. The incidence of malformations was lower than in the control group at 460 mg/kg bw/day (p<0.01 according to the Chi square test) and at 615 mg/kg bw/day. The number of fetus with malformations was comparable to control in the
800 mg/kg bw/day group.

Variations, malformations:
Statistically significantly higher incidence (p<0.05) according to the Chi square test was seen in the 615 mg/kg bw/day group only for the number of affected fetuses with 1 or more bones incompletely ossified. If only fetuses with 3 or more incompletely ossified bones were evaluated no statistically significant differences were observed. This variation was at a similar level in the 800 mg/kg bw/day group however statistics revealed no significance.
Also, in case of less ossified metacarpal/metatarsal, the number of affected fetuses and litters was statistically significantly higher (p<0.01 and p<0.05 respectively) according to the Chi square test in the 615 mg/kg bw/day group but not in the 800 mg/kg bw/day dose group. The incidences were not statistically significant when the litter means were compared. There were slightly higher incidences in case of some variations (retarded skull bones and unossified hyoid at 800 mg/kg bw/day, ossified sternebra and incomplete ossification of vertebra (SII or from SII) at 615 and 800 mg/kg bw/day), however without a statistical significance.
These increases were judged to be borderline cases considering the lack of clear statistical significances and the slight differences/low incidences. The slight differences might be related to reduced body weight gain and food consumption between g.d. 8 and 14 in the 800 and 615 mg/kg bw/day groups.
Slightly bent scapula (variation) occurred only in the test item treated groups (with an incidence of 1, 3 and 1 in the 460, 615 and 800 mg/kg bw/day groups, respectively) without a dose response. Two more fetuses (in one litter) had bent scapula (one with slightly thicker humerus) in the 800 mg/kg bw/day group, which was considered as a malformation. It should be noted, that those two fetuses belonged to the changed placentas, as detailed under external examinations.
The incidence of slightly bent or bent scapula as well as malformed humerus was low and may occur in control fetuses according to the historical control data, hence a test item response was not proved.

There was no indication on a test item effect in the 460 mg/kg bw/day group.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 137, 121, 105 and 115 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. There were no statistically significant increases in the incidence of visceral variations and malformations.

Variations:
Slightly dilated ventricles were recorded in the control and 615 mg/kg bw/day group in a few fetuses. However, the number of fetuses with the dilated ventricles was highest in the control group and no dose- response relationship was observed. Slightly enlarged peri-meningeal space was observed in one fetus in the 460 mg/kg bw/day group. Bilateral hydroureter were found in all groups without a dose-response. Hydroureter accompanied with dilated renal pelvis was observed in all groups without a statistically significant increase. A slightly malpositioned kidney was found in one control fetus.
Considering the lack of dose- response and the low incidence the above variations were not attributed to the treatment.

Malformations
Brain malformations were found in one fetus each of the mid and high dose group. One fetus in the 800 mg/kg bw/day group had external hydrocephaly and a hole in the thalamus region was found in cased of one fetus in the 615 mg/kg bw/day group.
Taking into account that external hydrocephaly and partial deficiency of brain tissue may occur in control fetuses with low incidences according to the background data of this laboratory, and the low incidences (one each at 615 and at 800 mg/kg bw/day group), the occurrence of these malformations was considered unrelated to the treatment with the test item.

Other effects:

no effects observed

Description (incidence and severity):

Fetal Examination (External, Visceral, Skeletal) - Overall
The number of evaluated litters/fetuses was 23/266, 21/243, 19/209 and 20/229 in the control, 460, 615 and 800 mg/kg bw/day groups, respectively.
There were 4/23, 0/21, 2/19 and 5/20 litters with malformed fetuses respectively. There were no statistically significant differences in the incidence of malformations over all dosing groups.

Effect levels (fetuses)

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 test item related adverse effect on the fetuses were observed.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

615 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as to be 460 mg/kg bw/day for maternal toxicity and and 615 mg/kg bw/day for overall developmental toxicity including teratogenicity.

Executive summary:

The test item was examined for its possible prenatal developmental toxicity in a study according to OECD 414. Groups of 28 to 30 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 460, 615 and 800 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 28 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 1 and 280 mg/g. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 91 and 105 % of nominal concentrations at both analytical occasions confirming proper dosing. 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 was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. 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. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. 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 23, 21, 19 and 20 evaluated litters in the control, 460, 615 and 800 mg/kg bw/day groups, respectively. One female died in the course of the study in the 800 mg/kg bw/day group due to an effect of the test item after clinical signs such as hunched back, piloerection and reduced activity and digging in the bedding in the days before. Necropsy revealed macroscopic changes in the lungs, liver and ileum. Histopathology supported these findings and additionally there were lesions in the heart observed. Similar test item related clinical signs were observed in the 800 and 615 mg/kg bw/day group. Reduced food consumption and lower body weight gain (including corrected body weight gain in the 800 mg/kg bw/day dose group) was observed in the 800 and 615 mg/kg bw/day group which was attributed to an effect of the test item. Slight reductions in the food consumption of the 460 mg/kg bw/day group animals were not considered as adverse. There were no significant differences in the body weight (including corrected body weight) of the dams. Lower values compared to control were measured in all dose groups (in similar manner) for the free thyroid hormone level FT4. The FT3 values were also statistically significantly lower in the 615 and 460 mg/kg bw/day groups. In the absence of a dose-response relationship these findings are considered not test-item related. The TSH hormone levels were similar in all groups. There were no test item related differences in thyroid weight among the dosing groups. The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Slight increase of post-implantation loss (early and late resorptions) including the occurrence of total post-implantation loss in one female both in the 800 and 615 mg/kg bw/day group were likely attributed to an effect of the test item, possibly due to maternal toxicity. The fetal weight was slightly lower and the incidence of growth retarded fetuses was higher in the 800 and 615 mg/kg bw/day groups (at a similar level), however statistical analysis revealed no significance. Hence, a test item effect was not proved. There were no significant differences in the ano-genital distance and placental weight parameters. At fetal examinations, there were no significant differences in the incidence of malformations at any dose level. There were no malformed fetuses found on external examination. In one litter, the placentas were observed as larger and or with wider margin in the 800 mg/kg bw/day group. Considering the lack of dose response or the low incidences, the visceral variations observed were not attributed to treatment. Two single cases of brain malformations such as an external hydrocephaly and a hole in the thalamus region in the dose groups of 800 and one 615 mg/kg bw/day group were considered incidental and toxicologically not relevant. Some increases in delayed ossifications might be related to maternal toxicity in the 800 and 615 mg/kg bw/day groups. Slightly bent scapula occurred in the test item treated groups with low incidences (1 to 3 fetuses per dose group) and two fetuses of one litter had bent scapula (one with slightly thicker humerus) in the 800 mg/kg bw/day group (latter in the litter with placenta alterations). Slightly bent or bent scapula as well as malformed humerus may occur in control fetuses according to the background data, hence a test item response was not proved.

In conclusion the oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose level of 800 mg/kg bw/day caused test-item related death of one animal. Clinical signs of animals in the 800 and 615 mg/kg bw/day groups were attributed to the treatment. The treatment with the test item caused reduced food consumption and body weight gain in the 615 and 800 mg/kg bw/day groups. Besides a significantly decreased corrected body weight gain in the 800 mg/kg bw/day group no significant differences in the body weight/corrected body weight values were detected. Thyroid-related parameters (FT3, FT4 TSH, weight, histopathology) were not affected by treatment. The slightly but statistically significantly increased postimplantation loss in the 615 and 800 mg/kg bw/day group is considered to be treatment-related. Although the incidence of body weight retarded fetuses was slightly increased in the mid and high dose group, there were no statistically significant differences in the mean body weight of the fetuses. No statistically significant differences in the incidence of malformations over all dosing groups were observed. Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as to be 460 mg/kg bw/day for maternal toxicity and and 615 mg/kg bw/day for overall developmental toxicity including teratogenicity.