2-ethylhexanoic acid, molybdenum salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted under GLP

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

publication

Title:

Developmental toxicity study of sodium molybdate dihydrate administered in the diet to Sprague Dawley rats

Author:

Murray, F.J. et al.

Year:

2014

Bibliographic source:

Reproductive Toxicology, Volume 49, November 2014, Pages 202–208, DOI: 10.1016/j.reprotox.2014.09.001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2001-01-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

disodium molybdate dihydrate

IUPAC Name:

disodium molybdate dihydrate

Constituent 2

Reference substance name:

Disodium molybdate

EC Number:

231-551-7

EC Name:

Disodium molybdate

Cas Number:

7631-95-0

Molecular formula:

Na2MoO4

IUPAC Name:

disodium tetraoxomolybdate

Constituent 3

Reference substance name:

10102-40-6

Cas Number:

10102-40-6

IUPAC Name:

10102-40-6

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Sodium molybdate dihydrate (SMD)
- Supplier: Climax Molybdenum, Phoenix, Arizona
- Physical state: white crystals
- Storage condition of test material: ambient, closed container, dry

Test animals

Species:

rat

Strain:

other: Crl: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: ~ 8 to 10 weeks upon arrival on gestational day 0 or 1
- Weight at study initiation: ~ 200-250 g on gestational day 3 (day of randomization into groups)
- Housing: solid-bottom, polycarbonate caging; acclimation and gestation: singly; enrichment: nestlets; bedding: Sani-Chips® cage litter (P.J. Murphy Forest Products, Montville, NJ)
- Diet (ad libitum): Purina Certified Rodent Chow No. 5002 (Purina Mills, Inc., Richmond, IN)
- Water (ad libitum): tap water
- Acclimation period: at least 3 days

All animals were used in compliance with the NRC Guide for the Care and Use of Laboratory Animals (2011).

ENVIRONMENTAL CONDITIONS
- Temperature: 21.7 - 22.8 °C
- Relative humidity: 35.82 to 55.44
- Air changes: 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
A premix was prepared by mixing chemical over a small portion of blank feed. The formulation was prepared by layering additional blank feed, the premix, and more blank feed in a twin-shell V-blender and mixing for approximately 15 minutes.
One dietary formulation for each group was prepared once.
The formulations were stored at ambient temperature (20°C). The dosed feed in the jars was changed at least once per week during conduct of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test chemical in the dosed feed formulations were conducted. Homogeneity of the dosed feed formulations was evaluated using the same batch size as required for the animal study and at the lowest and highest proposed dietary concentrations. Samples for analysis were collected from left, right, and bottom blender ports for each formulation. A sample of the blank feed was also sent for analysis.
The frequency of analysis was as follows:
- dose concentration analysis for all formulations (once), prior to study start.
- homogeneity once on the dose formulations for low and high dietary concentrations, prior to study start.
- stability once on the high and low dietary dose formulations out to 28 days, begun prior to study start (so the diets are never used beyond their confirmed stability).

Results:
The analyses indicated that the high and low dose formulations were stable for at least 28 days at room temperature. Analyses of aliquots of all dosing formulations also determined that the dietary dose formulations of sodium molybdenum dihydrate in the feed at 0, 100, 338, 675 and 1350 ppm (parts per million) were accurate; all dietary samples were all well within ± 10% of the target concentrations of molybdenum, and homogenous. The concentration of sodiummolybdate dihydrate added to the diet of 0, 100, 338, 675 and 1350 ppm are equivalent to molybdenum concentrations of 0, 40, 134, 268 and 536 ppm. The analyses of molybdenum by Michigan State University of the feed samples, gave values of molybdenum of 1.75 to 1.80, 39.7-40.8, 134-139, 264-268, and 540-542 ppm for the 0, 40, 134, 268 and 536 ppm feeds respectively The dose formulations were therefore appropriate for use and were used in this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (mated at the vendor over 4 days)
- Proof of pregnancy: a positive vaginal smear was designated gestational day (GD) 0.

Duration of treatment / exposure:

Gestational days 6 through 20

Frequency of treatment:

ad libitum (7 days/week)

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

concentration based on molybdenum in diet

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

concentration based on molybdenum in diet

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

concentration based on molybdenum in diet

Doses / concentrations 4

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

concentration based on molybdenum in diet

Doses / concentrations 5

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

concentration based on molybdenum in diet

No. of animals per sex per dose:

25 timed-mated (presumed pregnant) females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: in a dose-range finding study conducted previously, administration of sodium molybdate dihydrate in the diet, available ad libitum to pregnant rats from gestational day 6 to term necropsy on gestational day 20 was not associated with any treatment- or dose-related maternal findings at any molybdenum dose (0, 37.5, 187, 375 and 750 ppm of sodium molybdenum dihydrate and equivalent to at 0, 1-20 mg/kg/day molybdenum) or any time during gestation or at scheduled necropsy, including no effects on maternal body weights, weight gains, feed consumption in g/day or g/kg/day, clinical observations, pregnancy indices or organ weights. There were also no treatment- or dose-related developmental toxicity findings at any dose, including no effects on pre- or postimplantation loss, foetal numbers, sex ratio, body weights, or foetal external malformations or variations. There were no foetal external malformations or variations found in this study; this is comparable to the laboratories historical control database which indicates 17 foetuses with external malformations/variations out of 14,352 foetuses, approximately 0.12%.
Therefore, dietary concentrations of 0, 100, 338, 675, and 1350 ppm (equal to 0, 3, 10, 20 and 40 mg/kg bw/day of Mo) were chosen for the definitive developmental toxicity study.
- Rationale for animal assignment: timed-mated females were assigned to treatment groups in Provantis™ by stratified randomization, by body weight on gestational day 3, to provide uniform mean body weights across dose groups (± 20g).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, twice per day, at least 6 hours apart
- Cage side observations: morbidity/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations (out of cage) were conducted and recorded at least once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: gestational days 3, 6, 9, 12, 15, 18 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured on gestational days 3, 6, 9, 12, 15, 18 and 20.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20 (euthanasia by CO2 asphyxiation)
Identification and retention of any maternal gross lesions in 10% formalin.

ORGAN WEIGHTS: Yes
- Organs: uterus, liver, kidneys

ANALYTICAL CHEMISTRY:
- Serum from 10 arbitrarily selected females per group collected via cardiac puncture.
- Maternal livers and kidneys were arbitrarily chosen from 2 or 3 females per dose group on each mating day to equal 10 pregnant females per group. Foetal placentae grouped by litter were collected from these same 10 females/group

HISTOPATHOLOGY: Yes
The remaining maternal livers and kidneys/group were fixed in 10% neutral buffered formalin for subsequent histopathology of the 15 remaining pregnant females at 0 ppm and of the 15 remaining pregnant females at 1350 ppm.

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
- Number of full resorptions: Yes
- Number of uterine implantation sites (live and dead): Yes

Fetal examinations:

Live fetuses were dissected from the uterus after maternal termination on gestational day 20 and euthanised.
- Foetal weight and sex: all live foetuses
- External examinations: Yes, 100% live foetuses
- Soft tissue examinations: Yes, 50% live foetuses (decapitated)
- Skeletal examinations: Yes, 50% (intact fetuses)
- Head examinations: Yes, 50% (same fetuses which received visceral examination)

Statistics:

All statistical analyses were performed using Provantis™ software. For all statistical tests, p<0.05 was used as the criterion for significance.
Quantitative continuous data (e.g., maternal body weights and weight gains, feed consumption in g/kg and g/kg body weight/day) were subjected to the Provantis™ generalized ANOVA/ANCOVA test. This decision tree includes analysis of variance (ANOVA) and covariance, nonparametric analysis of variance, pairwise tests (Dunnett, 1955; 1964) for parametric and nonparametric data, and Levene’s test (Levene, 1960) for homogeneity of variance. For each variable analyzed, where there was evidence of difference between groups, the methodology also identified those groups which differ from the control group.
The uterine weight and uterine implant data (i.e., data presented in the Uterine Implantation Data Table) were subjected to the Kruskal-Wallis nonparametric analysis of variance, which is the default technique used by Provantis™ (Kruskal and Wallis, 1952; Conover, 1971). When there was evidence of a significant group effect, pairwise comparisons of each treated group with the control group were performed using Dunnett’s test on the ranks.
The foetal weight and sex ratio (i.e., data presented in the Group Mean Caesarean-Fetal Data Table) were subjected to a 1-way mixed ANOVA, which is the default technique used by Provantis™. When there was evidence of a significant group effect, pairwise comparisons of each treated group with the control group were performed using Dunnett’s test on group least square means. No statistical tests were performed on incidences of foetal malformations or variations.

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: Although no clear toxic effects were observed in either the dams or on the embryos/fetuses, the top dose is equivalent to approximately 20,000 times the average human daily intake of molybdenum from food and water of about 2 µg/kg bw/day.

Details on maternal toxic effects:
There were no treatment or dose-related effects on maternal body weights, weight changes, feed consumption in grams/day or grams/kg, body weight/day, or on maternal clinical observations, pregnancy indices, or maternal organ weights at any dose. There were also no biological or statistical differences among groups for the numbers of ovarian corpora lutea/female, for uterine implantation sites, or for uterine implantation losses per female at any dose.
In addition, there were no potential test article-related histopathological lesions identified during the pathology examination (kidney and liver).

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

> 40 mg/kg bw/day

Based on:

element

Remarks:

Mo

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no biological or statistical differences among groups for the numbers of foetuses, foetal sex ratios, foetal body weights, foetal external, visceral or skeletal malformations or variations per female at any dose. The incidences of the few foetal malformations and the more common foetal variations observed in the study were comparable to the historical control database of the laboratory on this rat strain and supplier. The foetal effects in this study also did not exhibit any treatment- or dose- related pattern of increased incidences and/or severities.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

> 40 mg/kg bw/day

Based on:

element

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis of the elements in blood and tissues showed that there were significant and dose related increases in molybdenum and copper in blood and all tissues examined.

Applicant's summary and conclusion

Conclusions:

NOAEL> approximately 40 mg/kg bw/day molybdenum (maternal toxicity).
NOAEL> approximately 40 mg/kg bw/day molybdenum (developmental toxicity).
Based on the results of this main developmental toxicity study (and the previous dose-range finding study) of dietary molybdenum, an essential element, it has no adverse effect on maternal or embryofoetal endpoints in rats up to and including 1350 ppm in the feed, corresponding to approximately 40 mg Mo/kg/day. Although no clear toxic effects were observed in either the dams or on the embryos/fetuses, the top dose is equivalent to approximately 20,000 times the average daily intake of molybdenum from food and water of about 2 µg/kg bw/day.