mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 July 1991 to 31 July 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

1984

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 87/302/EEC of Nov. 18, 1987 adapting to technical progress for the ninth time; Council Directive 67/548/EEC, pp. 24 - 26".

Version / remarks:

1988

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Test material form:

solid: particulate/powder

Remarks:

Powder / light beige

Details on test material:

- Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Chbb:THOM (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: The female rats were about 53 or 56 days old when supplied. At the beginning of the study (day 0, detection of sperm), the rats were 61 or 66 days old.
- Weight at study initiation: Their mean weight was approx. 222.8 g.
- Housing: During the study period, the rats were housed singly in type DK III stainless steel wire mesh cages.
- Diet: Ad libitum throughout the study (from the day of supply to the day of necropsy).
- Water: Ad libitum throughout the study (from the day of supply to the day of necropsy).
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % aqueous CMC solution.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each day the test material suspensions were freshly prepared shortly before the test material was administered. For the preparation of the suspensions, an appropriate amount of the test material was weighed and subsequently suspended in a 0.5 % aqueous carboxymethyl cellulose solution using a high speed sonicator. A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.
Due to technical reasons, the study was carried out in 2 sections. Each dose group was represented in each section. A treatment interval of 2 days elapsed before the next section.

VEHICLE
- Concentration in vehicle: 0, 200, 500 and 1 000 mg/100 mL.
- Amount of vehicle: 10 mL/kg The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determinations of the purity and the homogeneity of the test material itself were carried out before the beginning of the study (method: HPLC).
A reanalysis of the test material for verification of the stability will be carried out after completion of the current series of studies with this batch of test material.

Analyses of the suspensions of test material:
Analytical verifications of the stability of the test material suspensions up to 24 hours were carried out before the beginning of the study.
The stability of the test material suspensions was checked in the range-finding study in a comparable batch. Samples of the test material 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 (20 and 100 mg/kg bw/day). Six 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. The test material suspensions were analysed by HPLC.

Measurement Method
Reverse-phase-HPLC with UV-detection.
The concentration control analyses yielded mean concentrations of 94.4 % and 99.2 % for the 500 mg/100 mL, 96.4 % for the 200 mg/100 mL and 103.4 % for the 1 000 mg/100 mL samples.
The analyses of the homogeneity yielded mean concentrations and standard deviations of 102.8 % (± 5.15 %) and 92.4 % (± 2.68 % for the 200 and 1 000 mg/100 mL samples respectively.
The concentration control analyses confirm the theoretical values, the CMC-suspension is mixed homogeneousely.

Results of analyses of the test material:
The homogeneity of the test material was proven. The content of active ingredient was determined twice before the beginning of the study. The results of these determinations showed a purity of 92.7 % and 92.2 %.

Results of analyses of the suspensions of test material:
The stability of the test material suspensions over a period of up to 24 hours at room temperature was demonstrated.
The results of the analyses of the suspensions of test material confirmed the correctness of the prepared concentrations and the homogeneous distribution of the test material in the vehicle.

Details on mating procedure:

- Impregnation procedure: Cohoused
If cohoused:
- M/F ratio per cage: Four untreated female rats were mated with one untreated fertile male animal of the same breed.
- Verification of same strain and source of both sexes: Animals were of the same breed.
- Proof of pregnancy: If sperm were detected microscopically in the vaginal smear the animals were considered to be fertilised. This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).

Duration of treatment / exposure:

Once a day during the period of major organogenesis (day 6 to day 15 p.c.) always at approx. the same time of day (in the morning).

Frequency of treatment:

Daily

Duration of test:

Day 6 to day 15 p.c.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of doses for the present examination was based on the results of several preceding studies, in particular a range-finding study.
In a range-finding study, the test material was administered to pregnant rats (10/dose) by gavage from day 6 to day 15 post coitum at doses of 0, 50, 100 and 150 mg/kg bw/day. For a clear assessment of test material-related effects on the dams, the animals were sacrificed on day 16 p.c. Just before sacrifice, blood samples were taken for clinico-chemical and haematological examinations. Furthermore, liver and kidneys were weighed. Due to the sacrifice of the dams on day 16 p.c., only limited information on embryonic and foetal toxicity could be obtained.
150 mg/kg bw/day:
- clearly reduced food consumption between days 6 - 13 p.c.;
- statistically significantly lower body weights than in the control group between days 13 - 16 p.c.;
- body weight loss between days 6 - 8 p.c., reduced body weight gains on the following days;
- drastically reduced gravid uterine weights;
- drastically reduced corrected body weight gain;
- statistically significant reduction of erythrocytes, haemoglobin, haematocrit and platelets;
- significant increase in alanine aminotransferase and alkaline phosphatase;
- increased chloride, creatinine, urea and glucose values;
- decreased total protein, globulin, triglyceride and cholesterol values statistically;
- significantly increased absolute and relative liver weights (13 or 20 % higher than the relevant control values);
- statistically significant increase of the relative kidney weights (6 %)
- clearly increased post-implantation loss due to the increase a number of early and late resorptions;
- reduced number of live foetuses/dam;
- decreased mean foetal body weights.

100 mg/kg bw/day:
- clearly reduced food consumption between days 6 - 10 p.c.;
- reduced body weight gains on days 6 - 8 p.c.;
- marginally reduced corrected body weight gain;
- statistically significant reduction of erythrocytes, haemoglobin, haematocrit and platelets;
- increase in alanine aminotransferase (trend) and statistically significantly increased alkaline phosphatase values;
- increased glucose values;
- decreased total protein, globulin, triglyceride and cholesterol values;
- increased absolute (trend) and statistically significantly increased relative liver weights (10 or 13 % higher than the relevant control values);
- slightly increased post-implantation loss.

50 mg/kg bw/day:
- marginally reduced food consumption between days 6 - 8 p.c.;
- marginally reduced corrected body weight gain [questionable substance effect];
- statistically significant reduction of haemoglobin and haematocrit values;
- statistically significantly decreased cholesterol values.

Taking into consideration the results of this study with administration of the test material the following doses were fixed for the full-scale prenatal toxicity study in rats:
20 mg/kg bw: As the expected no observed adverse effect level (NOAEL).
50 mg/kg bw: As a dose which might be a minimal toxic effect level for dams and/or foetuses.
100 mg/kg bw: As the dose level with maternally toxic effects at which findings in foetuses may also be obtained.

- Rationale for animal assignment: On day 0, the animals were assigned to the different test groups according to a randomisation plan.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.).
- Cage side observations checked: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. 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 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.).

FOOD CONSUMPTION: Yes
- With the exception of day 0, the consumption of food was determined on the same days as was body weight.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes. On day 20 p.c., all females were sacrificed in a randomised order and examined macroscopically. The foetuses were dissected from the uterus and further investigated with different methods.
- Sacrifice on gestation day: On day 20 p.c., the dams were sacrificed in randomised order by cervical dislocation and the foetuses dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed.

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, including distribution of implantation sites.
- Number of early resorptions: Yes, early resorptions (only decidual or placental tissues visible or according to Salewski from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy).
- Number of late resorptions: Yes, embryonic or foetal tissue in addition to placental tissue visible.

Fetal examinations:

- External examinations: Yes: At necropsy each foetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all foetuses fixed in Bouin's solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a foetus, the foetus was finally sexed according to the appearance of its gonads.
Furthermore, the viability of the foetuses and the condition of the placentae, the umbilical cords, the foetal membranes and fluids were examined. Individual placental weights were recorded.
After these examinations, approximately one half of the foetuses per dam was placed in ethyl alcohol and the other half was placed in BOUIN's solution for fixation and further evaluation.
- Soft tissue examinations: Yes: After fixation in Bouin's solution, approximately one half of the foetuses of the dams of all groups was examined for any findings in the organs according to the method of Barrow and Taylor. After these examinations the relevant foetuses were discarded.
- Skeletal examinations: Yes: After fixation in ethyl alcohol, the skeletons of approximately one half of the foetuses were stained according to a modified method of Dawson. Thereafter, the skeletons of these foetuses were examined under a stereomicroscope. After these examinations the relevant foetuses were retained by litter.
- Head examinations: Not specified.

Evaluation criteria for assessing skeletons and organs of the foetuses:
There are differing opinions on the classification and assessment of changes in foetuses. Müntefering differentiates between malformations, which he defines as "severe morphological defects outside the range of variation of the species", and macroscopic/microscopic anomalies, which he defines as "slighter morphological deviations from normal". If these changes occur, they are regarded by Müntefering as a teratogenic effect. However, transition from variation to malformation is fluid, and the term "slighter morphological deviation" is not defined. Neubert, on the other hand, tends to describe a change as a morphological abnormality, anomaly or functional anomaly instead of malformation since malformation refers primarily to gross-pathological changes.
In the present investigations the following terms (definitions) were used for describing a change:
- Malformations (concerning external, soft tissue and skeletal observations): Rare and/or probably lethal changes were classified as malformations (e.g. exencephaly, atresia ani, hernia umbilicalis).
- Variations (concerning external, soft tissue and skeletal observations): Changes which occur regularly also in control groups and have generally no adverse effect on survival were regarded as variations (e.g. dilated renal pelvis).
- Retardations (concerning skeletal observations only): Delays in skeletal development compared with the norm at the time of the examination were considered to be retardations (e.g. sternebra(e) not ossified).
- Unclassified observations (concerning external and soft tissue observations, only): External or soft tissue observations, which could not be classified as malformations or variations (e.g. blood coagulum around placenta or urinary bladder).

Statistics:

The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft.

Examinations of dams and foetuses:
Dunnet'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 foetuses, weight of placentae, corpora lutea, implantations, pre- and post-implantation losses, resorptions and live foetuses.
Fisher's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all foetal findings.

Indices:

Calculations of conception rate and pre- and post-implantation losses were carried out.
The conception rate (in %) was calculated according to the following formula:

(number of pregnant animals / number of fertilised animals) x 100

The pre-implantation loss (in %) was calculated* according to the following formula:

((number of corpora lutea - number of implantations) / number of corpora lutea) x 100

The post- implantation loss (in %) was calculated* from the following formula:

((number of implantations - number of live foetuses) / number of implantations) x 100

* Calculation on the basis of each individual pregnant animal with scheduled sacrifice.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormal clinical findings in any dam of any one group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of the 100 mg/kg rats were slightly, but statistically significantly lower than the relevant control value on day 8 p.c. At the beginning of the treatment period (days 6 – 8 p.c.) the high dose dams (100 mg/kg bw/ day) lost some body weight; in the subsequent time, however, weight gain of the high dose dams sometimes reached or even exceeded (especially on days 8 - 10 p.c.) control values. If the body weight gain of the high dose dams during the whole treatment period, however, is calculated, these dams gained about 18 % less than the control dams.
Body weights and body weight gains of the dams of low and intermediate dose dams (20 mg/kg bw/day and 50 mg/kg bw/day) were similar to those of the controls. All observed differences between these groups are without any biological relevance.
The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) was statistically significantly reduced in high dose dams (100 mg/kg bw/day), which has to be assessed as a test material-related effect. All other differences between the groups are without any biological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the intermediate and high dose dams was statistically significantly reduced at the beginning of the treatment period (50 mg/kg group: days 6 - 8 p.c.; 100 mg/kg group: days 6 - 10 p.c.). The reduction in food intake (up to about 22 % less food consumption than the controls) of the high dose dams has to be assessed as being clearly related to the test material administration because body weights, body weight gain and corrected body weight gain of these animals were also impaired. The marginally reduced food consumption of the 50 mg/kg dams, which consumed up to about 9 % less food than the controls, however, is assessed as being a spontaneous effect, because it was not accompanied by statistically significant impairments of body weights/weight gains.
The food consumption of the low dose dams (20 mg/kg bw/day) did not show any differences of biological relevance if compared to the controls.
During the post treatment period (days 15 - 20 p.c.) food consumption of the high dose dams reached control values.

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):

The uterus weights of the animals were not influenced by the administration of the test material. The differences between the groups are without biological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related observations at necropsy in any of the dams. Oedema of the lungs were recorded for one control, one low dose and two high dose dams. A haemorrhagic liver was recorded for one control rat (No. 17) and one low dose rat (No. 31) showed a hydrometra; neither of these animals became pregnant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change.
Only pregnant dams with scheduled sacrifice (day 20 p.c.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.
In this study the following females were partially or totally excluded from the above mentioned calculations:
Control dams (0 mg/kg bw/day): Female No. 17 - not pregnant.
Low dose dams (20 mg/kg bw/day): Females Nos. 30, 31, 42, 43 and 47 - not pregnant.
Intermediate dose dams (50 mg/kg bw/day): Females Nos. 71 and 72 - not pregnant.
High dose dams (100 mg/kg bw/day): Females Nos. 86, 87, 88, 89 and 90 - not pregnant.

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the post-implantation losses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of resorptions and viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of resorptions. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam). The high number of live foetuses in the 100 mg/kg group, however, is the reason for the marginally, but statistically significantly lower mean foetal body weight and the increased number of skeletal retardations in this group.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 96 % (control group) and 80 % (20 and 100 mg/kg groups). There were no test material-related and/or statistically significant differences of biological relevance between the groups in conception rate. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam).

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam). The high number of live foetuses in the 100 mg/kg group, however, is the reason for the marginally, but statistically significantly lower mean foetal body weight and the increased number of skeletal retardations in this group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related and/or statistically significant differences of biological relevance between the groups in the number of viable foetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the incidental, relatively high number of live foetuses in the 100 mg/kg group, which is clearly higher than the corresponding control value (13.9 versus 12.8 live foetuses/dam) but is still fully within the historical control range (11.1 - 14.9 live foetuses/ dam).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the foetuses in the treatment groups (20, 50 and 100 mg/kg bw/day) was comparable with the control foetuses. The differences observed in comparison to the control are without any biological relevance.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Weight of foetuses: The mean foetal weights were not influenced by the test material administration. All values are within the range of biological variation. This includes the marginally, but statistically significantly lower foetal body weight in the 100 mg/kg group, which is caused by the increased number of live foetuses in this group.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An external malformation was recorded for one control foetus, only. For this foetus, an anasarca was noted. This malformation is also present in the historical control data. The external examination of the foetuses revealed no variations in any group. One so-called unclassified observation (placentae fused) was recorded for one foetus of test group 2 and one foetus of test group 3 (50 or 100 mg/kg bw/day).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Various malformations of the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asym.) or bipartite (asym.), lumbar vertebral body absent) were seen in 10 out of 158 (~ 6.3 %) foetuses (in 8 out of 24 litters (~ 33 %)) of the control, in 8 out of 141 (~ 5.7 %) foetuses (in 6 out of 20 litters (~ 30 %)) of the 20 mg/kg group, in 7 out of 155 (~ 4.5 %) foetuses (in 6 out of 23 litters (~ 26 %)) of the 50 mg/kg group and in 4 out of 144 (~ 2.8 %) foetuses (in 3 out of 20 litters (~ 15 %)) of the 100 mg/kg group. These differences were not statistically significant and did not show any dose-response relationship.
The variations elicited were related to the ribs (shortened 13th, accessory 14th ribs, rudimentary cervical or wavy ribs) and the sternum (sternebra(e) of irregular shape, bipartite, or accessory sternebra). Most of the skeletal variations recorded appeared without a clear dose-response relationship (e.g. sternebra(e) of irregular shape, sternebra(e) bipartite, 13th rib(s) shortened), can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance; the statistically significantly increased number of 100 mg/kg foetuses, however, which showed rudimentary cervical rib(s), has to be related to the oral administration of the test material to the dams.
In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches, sternebra(e), the skull and/or the hyoid bone) were found. In the 100 mg/kg group the number of foetuses with unossified sternebra(e) and the overall number of foetuses with skeletal retardations is statistically significantly increased. The foetal and litter incidence of "not ossified sternebra(e)" [17 % of the foetuses; 60 % of the litters] is just above the historical control range (3.4 - 16.0 % of the foetuses; 16.7 - 58.3 % of the litters). The increased number of high dose foetuses with retarded ossification, however, is not assessed as being test material-induced, but has to be related to the incidental, distinctly higher number of live foetuses/ dam in this test group in comparison to the control.
The foetal and litter incidences of skeletal retardations in test groups 1 and 2 (20 or 50 mg/kg body weight/day) do not show any statistically significant differences if compared to control values; the relevant values are fully in the range of the historical control.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the organs of the foetuses revealed no malformations in any of the groups. Variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant differences between the groups. All values are fully in the range of biological variation. No so-called unclassified observations (like blood coagulum around the placenta or urinary bladder) were recorded.

Other effects:

no effects observed

Description (incidence and severity):

Weight of placentae: The mean placental weights in the treatment groups (20, 50 or 100 mg/kg bw/day) were not influenced by the test material administration and were similar to the control values.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

(embryo-/foetotoxicity)

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Remarks on result:

other: No test material-induced teratogenic effects observed.

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

At 100 mg/kg bw/day marginal signs of embryo-/foetotoxicity in the form of an increased occurrence of foetuses with rudimentary cervical rib(s) were present, but no test material-induced teratogenic effects were seen up to and including the dose of 100 mg/kg bw/day.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Historical Control Data in Rats (Strain CHBB/THOM (SPF))

Skeletal Defect	Vehicle Controls N	Controls N	% of Total Malformations	% of Foetuses
Wavy ribs	6	22	9.78	0.04

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the no observed adverse effect level (NOAEL) on maternal and foetal rats is 50 mg/kg bw/day.

Executive summary:

The test material was assessed for its prenatal toxicity in Wistar rats according to OECD Test Guideline 414 and in compliance with GLP.

The test material was administered as an aqueous suspension to 20 - 24 pregnant female rats/group by stomach tube at doses of 20, 50 and 100 mg/kg bw on day 6 through day 15 post coitum (p.c.). A standard dose volume of 10 mL/kg body weight was used. The control group was dosed with the vehicle only (0.5 % aqueous carboxymethyl cellulose solution).

Food consumption and body weights of the animals were recorded regularly throughout the study period. 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 foetuses were dissected from the uterus, sexed, weighed and further investigated for any external, soft tissue and/or skeletal findings. The following findings were obtained and assessed as test material-related:

High dose dams (100 mg/kg bw/day):

- Statistically significantly reduced food consumption at the beginning of treatment period (days 6 - 10 p.c.);

- Statistically significantly lower mean body weights than the controls on day 8 p.c.; slight body weight loss on days 6 - 8 p.c. and, if calculated for the whole treatment period, a weight gain of about 18 % less than that of controls;

- Reduced corrected body weight gain;

- Statistically significantly increased number of foetuses with a skeletal variation (rudimentary cervical rib(s)).

Intermediate dose dams (50 mg/kg bw/day):

- No test material-related effects on dams or foetuses.

Low dose dams (20 mg/kg bw/day):

- No test material-related effects on dams or foetuses.

Thus, under the conditions of this full-scale study, the test material caused some overt signs of maternal toxicity and marginal signs of embryo-/foetotoxicity at 100 mg/kg bw/day. No test material-induced teratogenic effects were observed up to and including the dose of 100 mg/kg bw/day. There were no test material-induced, adverse effects on dams or foetuses of the intermediate or low dose groups (50 or 20 mg/kg bw/day).

In the preceding range-finding study, however, which was carried out under comparable study conditions (e.g. same batch, same rat strain, same treatment schedule), massive signs of maternal toxicity in the form of clearly reduced food consumption, retarded body weight gain, impairment of several haematological and clinicochemical parameters and/or increased liver and kidney weights were found at doses of 100 and 150 mg/kg bw/day, whereas marginal maternally toxic effects were still present at 50 mg/kg bw/day.

Under the conditions of the study the no observed adverse effect level (NOAEL) on maternal and foetal rats is 50 mg/kg bw/day.