Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to EPA OTS 798.4900. No data on GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Version / remarks:
(40 CFR 798.4900)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl ethyl ketoxime (MEKO)
- Source: Allied Signal, Inc. (Morristown, NJ, USA).
- Physical state: Liquid

Test animals

Species:
rat
Strain:
other: Sprague-Dawley CR:CD BE VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, NY, USA
- Age at study initiation: 90 days old
- Weight at study initiation: 222 - 291 g
- Housing: Individually in stainless steel cages
- Diet (e.g. ad libitum): Purina Certified Rodent Meal No. 5002
- Water (e.g. ad libitum): Fresh water, ad libitum
- Acclimation period: 13 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79 ºF
- Humidity (%): 64-70 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs ligth)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions were prepared fresh weekly in distilled water to provide dose levels as appropriate for use in either the dose range-finding studies or main studies.

VEHICLE
- Amount of vehicle (if gavage): Dose volumes were 10 mL/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Methyl ethyl ketoxime was analyzed for stability. Analysis of dosing solutions showed stability of the desired concentrations of methyl ethyl ketoxime for up to 14 days. Fresh dosing solutions were prepared weekly to adjust for body weight changes and administration of constant dose volumes. Analytical recoveries were within 6% of the respective nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- Proof of pregnancy: Evidence of mating was determined by the presence of a copulation (day 0 of day of gestation).
Duration of treatment / exposure:
Gestation Days 6-15.
Frequency of treatment:
Daily.
Duration of test:
20 days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 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: A preliminary dose range finding study was performed. Groups of six rats (dams) were given MEKO doses by gavage of 0, 25, 100, 200, and 400 mg/kg on gestation days 6–15. Methemoglobin determinations and reticulocyte counts were performed on all surviving dams on gestation days 7, 12, 16, and 20. Parameters evaluated were the same as in the main test (see below). Furthermore, blood samples were obtained from the orbital plexus of dams. MEKO doses for the main studies were chosen after evaluation of the results of the preliminary studies and the same duration of treatment during gestation was applied (see results included on section "Any other information on results").

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality and morbidity and for signs of toxicity (including behavioral abnormalities).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the treatment period, animals were observed between 1/2 and 2 h following dosing for sings of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were obtained on days 0, 6, 9, 12, 16, and 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: feed consumption was measured for gestation days 0–6, 9–12, 12–16, and 16–20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Dams that aborted were sacrificed at the time. Regular sacrifices were performed on gestation day 20 by carbon dioxide inhalation.

OTHER: Blood evaluations were not performed on the main study animals to avoid undue stress.
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: fetal sex ratios and fetal body weights
Fetal examinations:
- External examinations: Yes (litters and fetus)
- Soft tissue examinations: Yes (litters and fetus)
- Skeletal examinations: Yes (litters and fetus)
- Head examinations: No data

Fetuses were examined for external, internal (visceral), and skeletal abnormalities. Developmental malformations and variations were classified based on severity of anatomical changes and the extent of potential interference with organ and/or body functions. A fetal malformation was considered a permanent alteration that would adversely affect survival, growth, development, or functional competence. Fetal variations were considered to represent a delay in development, a transitory alteration or a permanent alteration not believed to adversely affect the survival, growth, development, or functional competence. Approximately 1/2 of fetuses were fixed in Bouin’s solution for subsequent visceral examination by the method of Wilson under low power microscope and 1/2 were eviscerated, fixed in 95% isopropyl alcohol, macerated in 1.5% aqueous potassium hydroxide, stained with Alizarin Red S, cleared in 25% aqueous glycerin and skeletal examinations performed under low power magnification.
Statistics:
All analyses were two-tailed with a minimum significance level of 5%. One way analysis of variance followed by Dunnett’s test was used to analyze maternal and fetal data including body weights, food consumption, and number of viable fetuses, implantation sites, and corpora lutea. Mann–Whitney U test was used to compare post implantation loss, dead fetuses, and resorption. Fetal sex ratios were analyzed using the Chi-Square test. Fisher’s Exact test was used to analyze the incidence and number of fetal malformations and variations utilizing the dam or dose (litter) as the experimental unit.
Historical control data:
(see results below)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent clinical signs of toxicity were observed at 200 and 600 mg/kg. Salient treatment-related findings were wobbly gait, general decreased responsiveness, urine stains, and apparent lack of urination control. In addition, weak body tone, coolness to the touch, salivation, and pale-appearing extremities were observed following dosing at the 600 mg/kg level. These clinical signs were generally transient and not observed prior to MEKO administration on the following day.
No treatment-related clinical signs were observed in the 60 mg/kg group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One rat of 60 mg/kg group died on gestation day 3 of an undetermined cause (prior to MEKO administration). The pregnancy status could not be determined. All other rats survived to scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean maternal body weights and body weight gains were similar between the control and 60 mg/kg groups throughout the study. In the 200 mg/kg group, mean body weights were similar to the control group. A significant reduction in body weight gain during gestation days 9–12 resulted in a significantly reduced weight gain for the entire treatment period (gestation days 6–16). At the 600 mg/kg level, significant body weight losses occurred during gestation days 6–9 and significantly reduced body weight gains were observed from gestation days 9–12. This resulted in a significant reduction in weight gain for the entire treatment period (when adjusted for gravid uterus weight). These changes also resulted in significantly lower mean body weights on gestation days 9, 12, 16, and 20 at this level. After the cessation of dosing, body weight gains were significantly increased indicating a recovery from treatment with the test article.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the 60 mg/kg dose level was similar to controls. In the other two dose groups, there were statistically significant reductions in feed consumption at varying intervals during MEKO administration which resulted in an overall decline. However, following cessation MEKO administration, feed consumption was similar to controls, but with an apparent increase on a gm/kg/day basis as a result of lower mean body weights.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At necropsy, enlarged spleens in all surviving dams at the 60, 200, and 600 mg/kg levels.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase in the mean number of early resorptions and post-implantation loss in the 600 mg/kg dose group was within the historical range of the laboratory
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase in the mean number of early resorptions and post-implantation loss in the 600 mg/kg dose group was within the historical range of the laboratory
Early or late resorptions:
no effects observed
Description (incidence and severity):
A slight increase (not statistically significant) in the mean number of early resorptions in the 600 mg/kg dose group was within the historical range of the laboratory.
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:
no effects observed
Description (incidence and severity):
One rat assigned to the 60 mg/kg group died on gestation day 3 of an undetermined cause (prior to MEKO administration). The pregnancy status could not be determined. All other rats survived to scheduled sacrifice with pregnancy rates of 100% in the 600 mg/kg group, 96% in the 200 mg/kg group, and 92% in the control and 60 mg/kg groups.
Other effects:
no effects observed
Details on maternal toxic effects:
There were no statistically significant diferences between the control and treated groups for any of the parameters of the cesarean section observations including the number of corpora lutea, implantation sites, viable fetuses, resorptions, fetal sex ratios, and fetal body weights

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
gross pathology

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: spleen

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control and treatment groups in the number of fetal body weights.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control and treatment groups in the number of fetal sex ratios.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related fetal malformations were noted. One fetus in the 200 mg/kg group was observed to have an omphalocele. All other fetuses in the control and treatment groups were observed to be externally normal.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were observed. Slight but not statistically significant increases in 7th cervical ribs and bent ribs were observed in the 600 mg/kg group, and 60, 200, and 600 mg/kg groups, respectively. Statistically significant decreases were observed in the number of litters at the 600 mg/kg level with sternebrae #5 and/or #6 unossified and reduced ossification of the 13th ribs (not considered biologically meaningful).
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No dose-related gestational effects, malformations or developmental variations at the highest dose level.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Preliminary Dose Range-Finding Study results:

All rats survived treatment. Pregnancy was found to be 100% except for 83.3% (5/6) at the 100 mg/kg level. No treatment-related changes were observed at 25, 100, and 200 mg/kg. Clinical signs of toxicity were at the 400 mg/kg level. These findings included wobbly gait, weak body tone, and general decreased responsiveness. The signs were transient and not observed prior to MEKO administration on the following day. Mean body weights and body weight gains in dams were generally comparable between the control, 25, 100, and 200 mg/kg levels. Slight body weight losses occurred at 400 mg/kg during gestation days 6–9. Dose-dependent increases in the percentage of reticulocytes and methemoglobin levels occurred in dams at all dose levels tested. The increases in reticulocytes were noted in dams at 200 and 400 mg/kg on gestation days 7, 12, 16, and 20 and at 25 mg/kg and 100 mg/kg on gestation days 12, 16, and 20. The increases in methemoglobin occurred at each interval tested at 25, 100, 200, and 400 mg/kg. At study termination, the methemoglobin levels were considerably lower than at the end of the treatment period indicating recovery was taking place. Necropsy findings included enlarged and blackish-purple spleens at 100, 200, and 400 mg/kg. Spleen adhesions were also observed in one animal at 400 mg/kg. All dams in the 25 mg/kg group were normal internally. All gestational parameters evaluated during Cesarean section observations including viable fetuses, early and late resorptions, fetal sex ratios, gravid uterus weights and fetal body weights were comparable between the control and treatment groups. No fetal external malformations or developmental variations were observed at any group.

Applicant's summary and conclusion

Conclusions:
NOAEL for developmental toxicity after a maternal oral exposure of methyl ethyl ketoxime was determined to be > 600 mg/kg bw/day in rats under test conditions.
Executive summary:

A Prenatal Developmental Toxicity Test was performed on methyl ethyl ketoxime in Sprague-Dawley rats according to EPA OTS 798.4900 Following preliminary dose range finding studies, groups of 25 pregnant rats were dosed by gavage with aqueous solutions of methyl ethyl ketoxime at 0 (control), 60, 200, or 600 mg/kg on gestation days 6–15. Several parameters were periodically observed, such as body weight, feed consumption, clinical pathology, ovaries and uterine content and fetal morphology. Dose dependent clinical signs of maternal toxicity including reduced body weight gains were noted at 200 and 600 mg/kg. At 60 mg/kg and above enlarged spleens were observed at necropsy. The preliminary study found methemoglobin formation and reticulocytosis indicative of anemia at these dose levels. No treatment-related gestational effects, malformations or developmental variations were observed in the rats. MEKO was not considered to have produced any treatment-related gestational effects and therefore, the NOAEL for developmental toxicity of methyl ethyl ketoxime was determined to be > 600 mg/kg bw/day in rats under test conditions.