Magnesium potassium titanium oxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-06-2009 to 03-07-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD and EU guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See remarks OECD 414

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See remarks OECD 414

Principles of method if other than guideline:

OECD 414: Due to a technical error no food consumption was available during Day 17-Day 20 for animal No. 35. Sufficient food consumption data was available for a proper evaluation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

436-900-9

EC Name:

-

Cas Number:

39290-90-9

Molecular formula:

Hill Empirical formula: K(0.2-0.7) Mg(0.4) Ti(1.6) O(3.7-3.95) CAS Empirical formula: K(0.2-0.7) Mg(0.4) Ti(1.6) O(3.7-3.95)

IUPAC Name:

Magnesium Potassium Titanium Oxide

Details on test material:

Batch: 0G99
White powder
Expiry date: 21 August 2001
Test substance storage: At room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: approximately 14 weeks.
- Weight at study initiation: 199-261 grams (day 0 post-coitum)
- Fasting period before study: not applicable
- Housing:
Pre-mating During acclimatization, females were housed in groups of 5 animals/cage in
Macrolon cages (MIV type, height 18 cm). During the weekend, mating procedures were suspended and the animals were housed in groups of maximum 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon
cages (MIII type, height 18 cm).
Post-coitum Females were individually housed in Macrolon cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to the start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-21.3
- Humidity (%): 34-79
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 02 June 2009 (start pairing) To: 23 June 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to
dosing and were homogenized to a visually acceptable level. No
adjustment was made for specific gravity of the test substance,
vehicle, and/or formulation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis (for accuracy and homogeneity) was done on one day during the treatment period (09 June 2009) according to a
validated method (NOTOX Project 490988). The analytical method for the quantitative analysis of the
test substance in formulation is based on total Titanium analysis
after acidic digestion. During this digestion, the test substance is
split into individual ions. The test substance cannot be analysed in
its original form. Stability of the test substance in formulations could
therefore not be tested.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: [yes ]
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
-After successful mating each pregnant female was caged: individually
-Any other deviations from standard protocol: no

Duration of treatment / exposure:

From day 6 to day 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

02 June 2009 (start pairing) to 03 July 2009 (end of in-life phase).

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of the dose range finding study (NOTOX Project 490450). Four groups of 6 females were exposed to 100, 300 and 1000 mg/kg/day for Days 6 to 19 post-coitum inclusive by oral gavage. There were no signs of toxicity.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations: Mortality/Viability


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards


BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3 and 6-20 (daily) post-coitum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, determined on Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Day 20 post-coitum
- Organs examined: External, thoracic and abdominal organs

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: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett,
1955) (many-to-one t-test) based on a pooled variance estimate was applied for the
comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be
assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
Mean litter proportions (percent per litter) of total fetal malformations and developmental
variations (external, visceral, skeletal and combined), and each particular external, visceral and
skeletal malformation or variation was subjected to the Kruskal-Wallis nonparametric ANOVA
test (Kruskal and Wallis 1952) to determine intergroup differences. If the ANOVA revealed
statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn 1964) was used to
compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance. Group means were calculated for continuous data and medians were calculated for
discrete data (scores) in the summary tables. Test statistics were calculated on the basis of
exact values for means and pooled variances.

Indices:

For each dose group reproduction parameters were expressed in two ways:
-As a mean (with standard deviation) of the number observed for each litter
-As a mean litter proportion calculated on a total group basis
For each litter the following calculations were performed:
Pre-implantation loss = (Number of corpora lutea - number of implantation sites) / Number of corpora lutea x 100
Post -implantation loss = (Number of implantation sites - number of live fetuses) / Number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given
finding both as the number of fetuses and the number of litters available for examination in the
group; and 2) considering the litter as the basic unit for comparison, calculating the number of
affected fetuses as a mean litter proportion on a total group basis.
Where:
Viable Fetuses Affected/Litter (%) = ( No. Viable Fetuses Affected/Litter) / No. Viable Fetuses/Litter x 100

Historical control data:

NOTOX Historical control data were used for external, visceral and skeletal malformations.

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 toxic effects observed.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no toxic effects observed.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal effects noted

Mortality:

Animal 42, Group 2 (100 mg/kg) was killed in extremis on Day 19 post coitum. Dark red discoloration and hardening of the uterus and cervical torsion were noted at the macroscopic examination; as this was an isolated finding, and in the absence of any dose-related relationship, these findings were not indicative of treatment-related toxicity.

Clinical signs:

Animal 42, Group 2 (100 mg/kg) had hunched posture, ptosis, piloerection, hypothermia and lethargy on Day 19 post coitum and was killed in extremis (see 7.2.1. Mortality above). Due to the isolated nature of these findings, seen only in one animal on a single day of the treatment period, they were likely due to chance and not indicative of treatment-related toxicity.

Incidental findings included alopecia of various body parts. This is not uncommonly seen in animals of this age and strain, and does not indicate treatment-related toxicity.

Fetal effects noted, external malformations and variations:

There were no test substance-related effects on fetal external morphology.

One fetus in the 1000 mg/kg group had a cyst-like growth from a cleft in the anterior palate into the buccal cavity. At visceral cephalic examination this fetus also showed anophthalmia (the right eye was absent) and misshapen brain. The isolated nature of these findings did not indicate any association with test substance treatment.

No external developmental malformations or variations were observed in any other fetuses in this study.

One fetus in the 100 mg/kg group and one fetus at 1000 mg/kg had their placenta grown together with an adjacent placenta. This finding was considered a pathological alteration and not a developmental alteration, and therefore was not classified as either a malformation or a developmental variation and was not included in the summary tables, but was included in the individual tables.

Visceral malformations and variations:

There were no test substance-related effects on fetal visceral morphology.

One fetus in the 1000 mg/kg group had external hydrocephaly (increase in space between the brain and the dura mater) and one fetus at 100 mg/kg had microphthalmia (left eye). Because each finding occurred in single fetuses and was seen in historical controls (hydrocephaly) or occurred in the lowest dose group (microphthalmia), they were not considered related to the test substance.

No other visceral malformations were observed in this study.

Visceral developmental variations observed in the test substance groups were accessory liver lobules, hemorrhagic adrenals, pale spleen and accessory spleen. All these variations occurred at similar frequencies in the control group and/or occurred infrequently.

One control fetus had a red area on the heart. This finding was considered a pathological alteration and not developmental alteration, and therefore was not classified as either a malformation or a developmental variation.

Skeletal malforations and variations:

There were no test substance-related effects on fetal skeletal morphology.

One control fetus had fused skull bones (the jugal was fused to the maxilla, unilateral). No other skeletal malformations were noted in this study. 

The mean litter proportion of bent ribs was 10.0%, 4.0%, 1.0% and 6.1% inthe control, 100, 300 and 1000 mg/kg groups, respectively. At 300 mg/kg, the mean litter proportion of bent ribs was significantly lower than in the control group and lower than the minimum historical control value (historical control data range: 6.7% - 15.2%). However, a decrease in the number of fetuses with bent ribs is not an adverse effect and the group distribution for this finding does not suggest a relation to test substance treatment. Therefore, the decreased number of fetuses with bent ribs at 300 mg/kg was considered to have arisen by chance.

Other skeletal developmental variations observed in the test substance groups were 14^thrudimentary ribs, ossified cervical centrum no. 1, slightly to moderately malaligned sternebra(e), reduced ossification of the skull, 7^thcervical rib(s), unossified metacarpals and metatarsals, unossified sternebra(e), 14^thfull ribs, 27 presacral vertebrae, generalized reduced ossification of the entire skeleton, unossified hyoid, unco-ossified and unossified vertebral centra. All these variations occurred at similar frequencies in the control group, occurred infrequently and/or in a manner that was not dose-related.

Applicant's summary and conclusion

Conclusions:

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Developmental findings
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study, the maternal
and developmental No Observed Adverse Effect Level (NOAEL) for TERRACESS P was
established as being at least 1000 mg/kg body weight/day.