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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 July 1991 - 27 August 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
November 1984
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-2-(4-chloro-2-methylphenoxy)propionic acid
EC Number:
240-539-0
EC Name:
(R)-2-(4-chloro-2-methylphenoxy)propionic acid
Cas Number:
16484-77-8
Molecular formula:
C10H11ClO3
IUPAC Name:
(2R)-2-(4-chloro-2-methylphenoxy)propanoic acid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 91-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance suspensions over a period of up to 24 hours at room temperature was demonstrated.









Test animals

Species:
rabbit
Strain:
Himalayan
Remarks:
Chbb:HM (outbred strain)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, FRG
- Age at study initiation: between 21 and 26 weeks old
- Weight at study initiation: mean 2.542 kg
- Housing: singly in type K 300/8 stainless steel wire mesh cages supplied by BECK ER & CO., Castrop-Rauxel, FRG
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data (fully air-conditioned rooms)
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous carboxymethyl cellulose solution (Tylose CB 30.000)
Details on exposure:
The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.).

PREPARATION OF DOSING SOLUTIONS: Each day the test substance suspensions were freshly prepared shortly before the test substance was administered. For the preparation of the suspensions, an appropriate amount of the test substance was weighed and subsequently suspended in a 0.5% aqueous carboxymethyl cellulose solution using a high speed sonicator (Ultra Turrax, JANKE & KUNKEL KG, FRG). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.

VEHICLE
- Justification for use and choice of vehicle: Carboxylmethyl cellulose (CMC)
- Concentration in vehicle: 0.5% aqueous CMC solution
- Amount of vehicle (if gavage): 10 mL/kg
- Purity: purified carboxylmethyl cellulose supplied by Hoechst AG (Tylose CB 30,000)
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance suspensions of the test substance for a period of at least 24 hours at room temperature were carried out for a similar batch in a prenatal range-finding study in rats. Samples of the test substance suspensions were sent to the analytical laboratories twice during the study period for verification of the concentrations. The samples which were sent for the first concentration control analysis toward the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (5 and 50 mg/kg body weight/day). 6 samples (2 from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running. Moreover retain samples of the test substance suspension (test group 50 mg/kg bw/day) were sent to the analytical laboratories for additional verification of the analytical result. The test substance suspensions were analyzed by HPLC.
Details on mating procedure:
After an acclimatization period, the does were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal®, trademark of HOECHST AG, Frankfurt) was injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study. The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).
Duration of treatment / exposure:
During the period of major organogenesis (day 7 to day 19 p.i.).
Frequency of treatment:
Once a day always at approx. the same time of day.
Duration of test:
On day 29 p.i., all surviving females were sacrificed in a randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods.
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Due to technical reasons, the study was carried out in 3 sections. Each dose group was represented in each section. A treatment interval of 7 days elapsed before the next section.
- Dose selection rationale: 5 mg/kg bw/day as the expected NOAEL, 20 mg/kg bw/day as a dose which might be a minimal toxic effect level for dams and/or fetuses, 50 mg/kg bw/day as the dose with maternally toxic effects at which findings in fetuses may also be obtained. The selection of doses for the present examination was based on the results of several previous studies.

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS: Yes
The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 29 p.i.). A check for mortality was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 29 p.i.).
BODY WEIGHT: Yes
All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i. The body weight change of the animals was calculated from these results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 29 p.i. minus weight of the uterus before it was opened minus body weight on day 7 p.i.).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The consumption of food was determined daily during the entire study period.
POST-MORTEM EXAMINATIONS: Yes
On day 29 p.i., the surviving dams were sacrificed in randomized order by intravenous injection of a pentobarbital and the fetuses were dissected from the uterus. Dams which died intercurrently as well as the contents of the uterus from these animals were investigated, if possible in the same way as at terminal sacrifice (exception: uterus weight).
After the dams had been sacrificed, they were necropsied and assessed by gross pathology.The uterus and the ovaries were removed and the following data were recorded:
- Weight of uterus before it was opened
- Number of corpora lutea
- Number and distribution of implantation sites classified as: live fetuses or dead implantations (early resoprtions, late resoprtions, dead fetuses)

Ovaries and uterine content:
The ovaries and uterine were removed and examined: Yes
Cesarean sections were performed on day 29 p.i.
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
- Number of dead and live fetuses: Yes
- Other: Individual placental weights were recorded.
Fetal examinations:
Examination of the fetuses after dissection from the uterus: At necropsy each fetus was weighed and examined macroscopically for any external findings. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded.
Soft tissue examination of the fetuses: After the fetuses had been sacrificed by CO2, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to assess the internal structure.
The sex of the fetuses was determined by internal examination of the gonads. If heads of fetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus, or cleft palate), the heads of these fetuses were severed from the trunk, fixed in BOUIN's solution and later processed and assessed according to WILSON's method. About 10 transverse sections were prepared per head. After the examination the heads treated in this way were discarded.
Skeletal examination of the fetuses: After the soft tissue examination all fetuses were placed in ethyl alcohol for staining of the skeletons (with the possible exception of the skulls) according to a modified method of DAWSON. The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed. After the examination the stained skeletons were retained by litter.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: animals with heads revealing severe findings
Statistics:
DUNNETT's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses.
FISHER's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Indices:
Calculations of conception rate (in %) and pre- and postimplantation losses (in %) were carried out.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One doe of test group 1 (5 mg/kg body weight/ day) died spontaneously on day 7 p.i. immediately after the first gavaging (but without any clear indications for misgavaging). Furthermore, two does showed minor skin lesions in the laryngeal area (50 mg/kg body weight/day). There were no abnormal clinical findings for any other does in the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One low dose dam was found dead on day 7 p.i. Although this female died immediately after the first gavaging, no clear indications for misgavaging, but some unspecific lung lesions were found at necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
All differences between the groups including the statistically significantly increased food consumption of the high dose group (50 mg/kg body weight/day) on days 27 - 28 p.i. (post treatment period) and the statistically significantly reduced food intake of the 5 and 20 mg/kg does on days 0 - 7 p.i. are assessed as being of spontaneous nature and without any biological significance.
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:
no effects observed
Description (incidence and severity):
All values for uteri weights lie within the range of biological variation. This includes the relatively low mean gravid uterus weight in the 50 mg/kg bw/day group, which is caused by an incidentally lower number of live fetuses/doe in comparison to the control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only some spontaneous necropsy findings were recorded for single animals of all dose groups including the controls. Most of these findings have to be related to the sacrifice of the animals (lungs with edema and/or marginal emphysema); moreover, agenesia of gallbladder (one control animal), lung with focal hemorrhages in one lobe (animal in 5 mg/kg body weight/day group) which died intercurrently), skin lesions (two animals in 50 mg/kg body weight/day group) and blind ending uterine horns (one animal in 50 mg/kg body weight/day group) were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight, statistically significant increase in the number of late resorptions at 50 mg/kg body weight/ day (see table 1). However, the total number of late resorptions in this group (5 of a total of 96 implantations) was small, and in the context of the overall postimplantation loss, was considered not to be associated with treatment. The respective values calculated for the high dose group are fully in the historical control range.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no signs of adverse maternal toxicity in highest dose level of 50 mg/kg bw/day

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not examined
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):
Only one type of external variation (pseudoankylosis) was found, which occurred without any relation to dosing. It was seen in single fetuses of all groups except test group which received 50 mg/kg body weight/day. There were no so-called unclassified observations (like placentae fused) in any of the fetuses.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations of the fetal skeletons were noted for 2 out of 97 control fetuses (in 1 out of 14 litters) and 1 out of 96 intermediate dose fetuses (in 1 out of 15 litters). These malformations were related to the vertebral column (cervical/thoracic vertebral body/bodies fused and/or of irregular shape) and the ribs (fused).The variations elicited were related to the skull (splitting of skull bones, epactal bone between nasal and frontal bones), the ribs (accessory 13th rib(s) and rudimentary cervical rib(s)), the vertebral column (accessory thoracic vertebra) and the sternum (sternebra(e) of irregular shape, fused or accessory sternebra). Most of these skeletal variations occurred without a clear dose-response relationship and/or without any biologically relevant, statistically significant differences between the groups; however, the occurrence of fused or irregular-shaped sternebra(e) and consequently the fetal and litter incidence of overall skeletal variations was clearly increased in the 20 mg/kg bw/day group. Both findings were also found quite frequently in the historical control data. Therefore and because no dose-response relationship is present, the increased number of fetuses with skeletal variations in the intermediate dose group is assessed as being of spontaneous nature. In all groups signs of retardations (incomplete or missing ossification of skull bones, vertebral column and sternebra(e)) were found; they occurred in a comparable frequency in the control and the substance-treated groups. All differences between the groups concerning fetal skeletal retardations are without any biological relevance.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One control and one high dose fetus each showed soft tissue malformations. For the control fetus agenesia of gallbladder was observed, while the high dose fetus had a septal defect, which led also to a dilated aortic arch and a dilated aorta descendens. All soft tissue malformations present at a low incidence in the historical control data and are assessed as being of spontaneous nature. Variations were detected in each group including the control; all occurred without a clear dose-response relationship and/or can be found at a comparable incidence in the historical control data. Aside from a separated origin of carotids, a very common finding in the rabbit strain used, another soft tissue variation (heart with traces of interventricular foramen/septum membranaceum) was also found quite frequently.Furthermore, one fetus of the low and intermediate dose group each (5 and 20 mg/kg body weight/day) exhibited hypoplasia of gallbladder and another one (20 mg/kg body weight/day) showed dilated renal pelvis. One control fetus showed a so-called unclassified observation (focal liver necrosis).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no indications for adverse developmental toxicity or teratogenicity found up to the highest dose tested (50 mg/kg bw/day)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of basic results

 

control

5 mg/kg bw/day

20 mg/kg bw/day

50 mg/kg bw/day

No. of inseminated rabbits

15

15

15

15

No. of pregnant rabbits

15

15

15

14

No. of implantations/rabbit

7.3

7.1

6.8

6.9

No. of late resorptions/rabbit

0.1

0.0

0.1

0.4 (a)

No. of live fetuses/rabbit

6.9

6.5

6.4

5.9

Mean fetal weight (g)

40.2

40.1

39.5

40.7

No. of fetuses with incomplete ossification of sternebrae

28

30

23

30

Significantly different from control: a = P<0.05; b= P< 0.01

 

Applicant's summary and conclusion

Conclusions:
The developmental toxicity study with MCPP-P acid (R-isomer, CAS 16484-77-8) in rabbits showed a NOAEL for maternal and developmental toxicity including teratogenicity 50 mg/kg bw/day, the highest dose level tested.
Executive summary:

A developmental toxicity study according to OECD TG 414 was conducted with 15 inseminated female Himalayan rabbits treated each daily orally by gavage with MCPP-P acid (R-isomer, CAS 16484-77-8). Doses of 0, 5, 20 and 50 mg/kg bw suspended test substance (vehicle: 0.5% aqueous carboxymethyl cellulose) was administered from day 7 through day 19 p.i. with a standard dose volume of 10 mL/kg. Food consumption and body weights of the animals were recorded regularly throughout the study period and the state of health of the animals was checked each day. On day 20 post coitum, all females were sacrificed and assessed by gross pathology. The fetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and/or skeletal findings. No signs of toxicity were elicited throughout the study period, neither for maternal nor for developmental toxicity up and including the highest dose tested. Based on the results of this full-scale prenatal toxicity study, the no observed adverse effect level (NOAEL) for maternal as well as developmental toxicity including teratogenicity is 50 mg/kg bw/day for MCPP-P acid (R-isomer) in rabbits.