kresoxim-methyl (ISO);methyl (E)-2-methoxyimino-[2-(o-tolyloxymethyl)phenyl]acetate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

20 days exposure during pregnancy

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japan/MAFF: Testing Guidelines for Toxicology Studies: Teratogenicity Study, pp. 48 - 49 (1985)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate

EC Number:

604-351-6

Cas Number:

143390-89-0

Molecular formula:

C18 H19 N O4

IUPAC Name:

methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate

Details on test material:

- Name of test material (as cited in study report): Reg. No. 242 009 (test substance number: 91/180-2)
- Lot/batch No.: N 36 (III C1); date of manufaturing: 1991-10-23
- Storage condition of test material: room temperature; in darkness
- Physical state: solid (powder) / light brown
- Analytical purity: 93.7% (Reversed-Phase - HPLC with UV-Detection)
- Stability under test conditions: the storage stability was guaranteed over the study period
- Other: the homogeneity of the test substance was confirmed by analysis (Reversed-Phase - HPLC with UV-Detection)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar rats (Chbb:THOM (SPF)) supplied by Karl THOMAE, Biberach an der Riss, FRG, which were free from clinical signs of disease, were used for the investigations
- Age at study initiation: sexually mature virgin (73 days old when provided, 81-83 days old at day 0, detection of sperm)
- Weight at study initiation: approx. 225 g at day 0
- Housing: during the study period, the rats were housed singly in desinfected type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area about 800 cm2 ).
- Diet (e.g. ad libitum): ground Kliba 343 feed rat/mouse/hamster supplied by KLINGENTALMUHLE AG, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): drinking water of tap water quality from water bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

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 weighted 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 (if other than water): solubility
- Concentration in vehicle: 0 mg/100 ml (in the control group), 1000 mg/100 ml (in the 100 mg/kg bw/day), 4000 mg/100 ml (in the 400 mg/kg bw/day group) and 10000 mg/100 ml (in the 1000 mg/kg bw/day group)
- Amount of vehicle (if gavage): 10 ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the aqueous test substance suspensions for a period of at least 24 hours at room temperature and its homogeneous distribution were carried out before the beginning of this full-scale study for similar batches (stability: batch No.: CP 5977; homogeneity: batch No.: CP 5864). Samples of the aqueous test substance suspensions were sent to the analytical laboratory twice during the study period for verification of the concentrations. The test substance suspensions were analyzed by HPLC.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: on the day 1 after acclimatization period mating took place from about 4 p.m. to about 7:30 a.m.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: if sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized. This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).

Duration of treatment / exposure:

the test substance was administered to the animals orally (by gavage) 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:

On day 20 p.c., all females were sacrificed in randomized order and examined macroscopically. The fetuses were dissected from the uterus and further investigated with different methods.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 400 and 1000 mg/kg bw/day
Basis:
actual ingested
gavage

No. of animals per sex per dose:

25 pregnant female animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based in a preliminary study where the test substance was administered to pregnant Wistar rats (max. 10/group) by gavage daily for a period of 10 days (days 6 - 15 post coitum) in doses of 400, 800 and 1,200 mg/kg body weight/day. For comparison, a group of sham treated (0.5% aqueous carboxymethyl cellulose) animals was used as a control.

The primary aim of this range-finding study was to find a dose which should induce "some overt maternal toxicity such as slight weight loss, but not more than 10% maternal deaths" and could be used as the highest dosage for the following full-scale prenatal toxicity study. Therefore, the dams were already sacrificed on the day following the last substance administration (i.e. day 16 p.c.). At necropsy the unopened uterus was weighed and the darns assessed by gross pathology. The fetuses were removed from the uterus, placental and fetal body weights were determined and the fetuses were subjected to a rough external examination.

The following findings were obtained and assessed as being possibly substance-related
- 1200 mg/kg body weight/day: no statistically significantly reduced food consumption at the beginning of the treatment period (days 6 - 8 p.c.)
- 800 mg/kg body weight/day: no substance-related effects on dams, gestational parameters or fetuses
- 400 mg/kg body weight/day: no substance-related effects on dams, gestational parameters or fetuses.

Taking into consideration the results of the above mentioned range-finding study, the following doses were fixed for the present full-scale prenatal toxicity study in Wistar rats with the test substance:
- 100 mg/kg bw: as the expected no observed adverse effect level
- 400 mg/kg bw: as the intermediate dose level
- 1000 mg/kg bw: as the dose level at which some maternally toxic effects and/or substance-induced findings in the fetuses could not be ruled out; testing at higher dose levels (> 1000 mg/kg body weight/day) were not considered to be necessary due to the recommendations given in the test guidelines mentioned below concerning the limit test.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check for mortality 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 in table were included.

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 (net maternal body weight change) 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
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; with the exception of day 0, the consumption of food was determined on the same days as was body weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.; the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus.
- Organs examined: 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: (1) weight of uterus before it was opened, (2) number of corpora lutea and (3) number and distribution of implantation sites classified as live fetuses or dead implantations ([a] early resorptions, only accidental 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; [b] late resorptions, embryonic or fetal tissue in addition to placental tissue visible; [c] dead fetuses, hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

For examinations of darns and fetuses the DUNNETT-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters: food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of pre-implantation loss, post-implantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight.
FISHER's Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
The WILCOXON-Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter.

Indices:

conception rate, pre- and post-implantation loss, malformations, variations, retardations, unclassified observations

Historical control data:

Historical control data of the testing laboratory were available and attached to the study report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no substance-related effects on the dams concerning food consumption, body weight, body weight change, uterine weights, corrected body weight change, clinical and necropsy observations up to and including a dose of 1000 mg/kg body weight/day. There were no differences of biological relevance between the control and the substance-treated groups (100, 400 and 1000 mg/kg body weight/day) in conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre- and the post-implantation losses.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No dose- and/or substance-related differences were recorded for placental and fetal body weights. The external, soft tissue and/or skeletal examination of the fetuses revealed no differences between the control and the substance-treated groups which might be related to the test substance administration. Number and type of the fetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations, recorded for the 100, 400 and 1000 mg/kg fetuses were substantially similar to concurrent and/or historical control values.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

3

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

1) Reproduction of dams:

The conception rate reached 96% in the control group, 92% in test groups 1 and 2 (100 or 400 mg/kg body weight/day) and 100% in the 1000 mg/kg group. There were no substance-related and/or biologically relevant differences between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the post-implantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

 

2) External examination of the fetuses:

The external examination revealed three fetuses with external malformations. For one low dose fetus anasarca and cleft palate was recorded. One intermediate dose fetus showed acaudia. One high dose fetus had a meningocele and a unilateral microphthalmia. No external malformations were observed in any of the control fetuses. These malformations or very similar ones are also present at a low incidence in the historical control.

The external examination of the fetuses revealed no variations in any group. One so-called unclassified observation (placenta (e) necrobiotic) was recorded for the one dead fetus of test group 3 (1000 mg/kg body weight/day). Necrobiotic placentae appear also occasionally in control animals and are not associated with treatment.

 

3) Soft tissue examination of the fetuses:

- The examination of the organs of the fetuses revealed only one soft tissue malformation in one high dose fetus. For this fetus hydrocephaly with dilatation of both lateral brain ventricles was noted. Hydrocephaly occurs also spontaneously in control fetuses of the rat strain used and is consequently present at a low incidence in the historical control data.

- Variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant and/or biologically relevant differences between the groups. Both findings are very common ones in the rat strain used and all respective values are fully in the range of biological variation.

- No so-called unclassified observations (like bloody inhibition of kidney(s)) were recorded during the soft tissue examination.

 

4) Skeletal examination of the fetuses:

-Various malformations of the sternum, the ribs and/or the vertebral column were seen in 5 out of 168 (=3.0%) fetuses (in 5 out of 24 litters (= 21%)) of the control, in 11 out of 167 (= 6.6%) fetuses (in 7 out of 23 litters (= 30%)) of the 100 mg/kg group, in 13 out of 156 (= 8.3%) fetuses (in 7 out of 22 litters (= 32%)) of the 400 mg/kg group and in 10 out of 172 (= 5.8%) fetuses (in 8 out of 25 litters (= 32%)) of the 1,000 mg/kg group. All skeletal malformations occurred without any statistically significant differences between the substance-treated groups and the concurrent control group and did not show a clear dose-response relationship. Furthermore, most of the aforementioned skeletal malformations or very similar ones can be found at a comparable fetal/litter incidence in the historical control data.

- The skeletal variations elicited were related to the ribs (shortened 13th, accessory 14th or rudimentary cervical rib(s)), the sternum (sternebra (e) of irregular shape or bipartite), the skull (epactal bone between parietal and interparietal bones) and the vertebral column (accessory thoracic or lumbar vertebra). All skeletal variations recorded appeared without a clear dose-response relationship, can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance.

- In all groups signs of skeletal retardation occurred substantiated by incomplete or missing ossification of skull (incl. hyoid) bones, vertebral column and sternebra (e).

 

The only statistically significant difference between the substance-treated groups and the concurrent control group concerning skeletal retardations was an increased occurrence of incompletely ossified thoracic vertebral body/bodies in the 1000 mg/kg group. If, however, the respective fetal (13%) and litter (56%) incidences and the mean percentage of affected fetuses/litter (14.2%) are compared with the historical control data it becomes obvious, that these values are fully within the historical control range (fetal incidence: 8.1% (0 - 49.1%); litter incidence: 22.8% (0 - 100%); mean percentage of affected fetuses/litter: 0 - 50%}.

 

Therefore, the statistically significantly increased occurrence of incompletely ossified thoracic vertebral body/bodies at 1000 mg/kg is finally considered to be spontaneous in nature. All other differences between the groups in respect to skeletal retardations appeared without statistical significance and/or without a clear dose-response relationship.