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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication/study report which meets basic scientific principles. The method is validated by ECVAM but is not available as a guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The potential embryotoxicity of a test substance is assessed in the Micromass test (limb bud micromass assay) by exposing undifferentiated rat embryo limb bud mesenchymal cells to the test substance and measuring the effect on specific parameters (cell differentiation, cell viability, cell number, neutral red uptake, cell growth) when the cells have differentiated into chondrocytes. The limb bud cells are isolated from rat embryos on gestation day 13 and a single cell suspention is divided into wells on a 96-well plate. Cell medium with or without the test substance is added and the plates are incubated for 5 days. The ID50 (50% inhibition of cell differentiation and number of foci) and IC50 (50% inhibition of cell viability and growth) are determined and used to classify the test substance as non-embryotoxic, weak embryotoxic or strong embryotoxic substance. The method used in the Micromass test (limb bud micromass assay) was based on established techniques (Flint, O. P., and Orton, T. C. (1984). An In Vitro Assay for Teratogens with Cultures of Rat Embryo Midbrain and Limb Bud Cells. Toxicol. Appl. Pharm. 76, 383-95) and similar to methodology validated by ECVAM (Micromass Test, method of Brown, INVITTOX No. 122) and available on the website (http://ecvam.jrc.it).
GLP compliance:
no
Remarks:
This non-guidleline ex vivo/in vitro study has been well conducted and reported. All raw data will be stored for 75 years in the archives of The Dow Chemical Company.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-amino-2-methyl-1,3-propanediol, AMPD, aminomethylpropanediol
- Analytical purity: 99.9%
- Lot/batch No.: WF114801I1
Specific details on test material used for the study:
- Test material: XU-12398.00
- Chemical name: 2-amino-2-methyl-1,3-propanediol
- Synonyms: AMPD, aminomethylpropanediol
- Supplier: ANGUS Chemical Company, Buffalo Grove, Illinois
- Lot #: WF114801I1
- Purity: non-GLP certificate of analysis lists the purity of the test material as 99.9% by weight.
- Molecular formula: C4H11NO2
- Molecular weight: 105.1
- CAS number: 115-69-5

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (CRL) Inc., Portage, Michigan, USA
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 200-250 g (females)
- Housing: the animals were housed one per cage in stainless steel cages with wire mesh floors, suspended above absorbent paper. Non-woven gauze were placed in the cages to provide a cushion from the flooring for rodent feet. The cages contained a feed crock and a pressure activated lixit valve-type watering system. Enrichment for the rats included the use of a non-woven gauze pad per animal in caging, a stainless steel object in the cage for manipulation, and periodic rotation of each rack of cages in the animal room to allow animals a variety of visual experiences.
- Diet: LabDiet Certified Rodent Diet #5002 in pelleted form (PMI Nutrition International, St. Louis, Missouri, USA), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 1 weeks prior to scheduled Cesarean section

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 (maximum permissible excursion was 22 ± 3 °C)
- Humidity (%): 40-70
- Air changes (per hr): 12-15, on average
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: not applicable
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
other: not applicable
Analytical verification of doses or concentrations:
no
Details on mating procedure:
The cells used for the in vitro assay were isolated from the embryos of time-mated rats.
Duration of treatment / exposure:
The embryo cells used in the in vitro assay were exposed constantly for 96 h. The rats, from which the embryos were derived, were not exposed to the test substance.
Control animals:
other: not applicable
Details on study design:
Range-finding study:
The first experiment was performed with concentrations up to 1000 µM, as this was approximately ten times higher than the upper estimate of the achieved steady state serum concentration following in vivo limit dosing (1000 mg/kg bw/day). As 50% cytotoxicity was achieved the same dose range was selected for the second experiment.

Procedure for initiating the cell culture:
The cells were isolated by euthanising the rats by carbon dioxide inhalation on GD 13 and isolating the embyos aseptically from the uterus in sterile Hank’s Balanced Salt Solution (HBSS). Hind limb buds were then removed by microdissection, pooled and dissociated using 1.0% trypsin + 0.1% Ethylenediaminetetraacetic acid (EDTA) in calcium/magnesium free phosphate buffered saline (CMF). The resulting cell suspension was rinsed with culture medium (10% v/v FCS in Ham’s F12), passed through a 100 µm pore size nylon mesh cell strainer to remove clumps of cells, and assessed for cell viability and number of cells using 0.1% trypan blue and a hemocytometer. The cells were then re-suspended in culture medium to achieve a final concentration of approximately 2 x 10E7 viable cells/mL.

Procedure for exposure of the cells:
Aliquots of final cell suspensions were seeded onto 24-well surface-modified polystyrene cell culture plates (PrimeraTM, BD, New Jersey, USA) at 10 µL per spot (200,000 viable cells) and incubated at 37 ºC in an atmosphere of 5% CO2/95% humidified air for two hours to allow for cell attachment. Following attachment, each well was gently flooded with 1 mL culture medium with or without test material. AMPD was added directly to the culture media (10% v/v fetal calf serum (FCS) in Ham’s F12) following solubilization in a separate vehicle when necessary (≤ 0.05% v/v DMSO for the positive control atRA). Test and positive control solutions were prepared in the appropriate vehicle with the highest concentration pH adjusted to 7.2-7.4 prior to serial dilution. The plates were then returned to the incubator for the duration of the 5-day (approximately 96 h) static culture.

Number of replications:
3 replicates were used per concentration level in two independent experiments.

Controls:
All-trans-retinoic acid (atRA) was used as the positive control at concentrations of 0.000033, 0.00033, 0.0033, 0.01, 0.033, 0.33, 1 and 33 µM, previously found to induce approximately 10-100% inhibition in chondrocyte differentiation. Negative controls with culture medium and DMSO were included in both assays.

Cytotoxicity (total number of viable cells):
The cell viability was determined after approximately 96 h of culture. The treated culture media was carefully removed and the cells rinsed two times with saline. Following fixation of the cells with 2.5% glutaraldehyde for 20 minutes, cell viability was assessed via cellular uptake of neutral red. The cells were cultured in the presence of 0.005% neutral red in fresh culture media for 2-3 hours followed by removal of the media, three rinses with saline and acid alcohol elution of the neutral red from the viable cells. The absorbance of the eluted neutral red were then read at 540 nm using a multi-well plate spectrophotometer.

Measurement of differentiated chondrocytes:
Following removal of the acid alcohol eluate, each well was gently rinsed with saline and incubated overnight at room temperature with 1% alcian blue in 0.1N hydrochloric acid, a dye which stains cells rich in anionic glycoconjugates such as chondrocytes. The alcian blue solution was then removed and each well was gently rinsed with saline. Assessment of chondrocyte differentiation was determined by (1) manual quantitation of the stained chondrocyte foci and (2) eluate absorbance measurement at 620 nm using a multi-well plate spectrophotometer following elution of the alcian blue stain using freshly prepared 6M guanidine hydrochloride.

Data analysis to determine cell viability and growth:
Dose response curves were plotted as a percent of the vehicle control from the measured results (neutral red absorbance or number of alcian blue positive foci or alcian blue absorbance). Using non-linear regression analysis (curve fitting), the IC50diff and IC50cyt were calculated. The ratio of the IC50cyt to IC50diff values was compared. Interpretation of a positive response was evaluated in the context of the data using the two-fold rule and a linear discrimination analysis prediction model as follows:
A) 2-fold rule: Embryotoxicity potential would be suggested if the IC50diff value was less than half the IC50cyt value (Uphill et al., 1990)
B) Prediction model: Linear discrimination algorithm analysis was used to predict one of three embryotoxicity potentials: non-, weak, or strongly embryotoxic.

Function I 6.65 * log (IC50diff) – 9.49
Function II 6.16 * log (IC50diff) – 8.29
Function III -1.31 * log (IC50diff) – 1.42

If the result of function I exceeded the results of function II and III, then the chemical would be predicted to be non-embryotoxic; if the result of function II exceeded the results of function I and III, the chemical would be predicted to be weakly embryotoxic; if the result of function III exceeded the results of functions I and II, the chemical would be predicted to be strongly embryotoxic (Genschow et al., 2000 and 2002).

Examinations

Maternal examinations:
General clinical observations. Open field examination.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
other: not applicable
Basis for effect level:
other:
Remarks on result:
other: AMPD was predicted to be non-embryotoxic in the rat embryo limb bud micromass assay.

Maternal abnormalities

Key result
Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Effect levels (fetuses)

Key result
Dose descriptor:
other:
Remarks on result:
other: AMPD was predicted to be non-embryotoxic in the rat embryo limb bud micromass assay.

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Cytotoxicity/ Cell viability:

Table 1: Results of cytotoxicity and chodrocyte differentiation, average of 2 trials

Concentration (µM)

Cytotoxicity

(% of control)

Chondrocyte differentiation, Foci Quantification method (% of control)

Chondrocyte differentiation, Alcian Blue Elution method (% of control)

AMPD

Mean ± SD

N

Mean ± SD

N

Mean ± SD

N

0.01

93.7 ± 13.6

6

92.5 ± 8.6

6

87.8 ± 15.7

6

0.1

84.1 ± 7.3

6

92.9 ± 8.6

6

92.5 ± 42.3

6

1.0

73.9 ± 20.9

6

84.4 ± 18.0

6

81.4 ± 22.4

6

10

77.0 ± 21.7

6

71.8 ± 19.4

5

81.8 ± 19.2

6

100

76.8 ± 17.4

6

69.4 ± 14.9

6

88.0 ± 25.9

6

1000

90.2 ± 42.5

6

81.3 ± 15.4

6

95.3 ± 10.2

6

 

IC50cyt= 1000µM / NQ

 

IC50diff= NQ

 

 

 

atRA

 

 

 

 

 

 

0.000033

100.5 ± 20.3

6

92.3 ± 14.0

6

67.5 ± 17.5 

6

0.00033

94.0 ± 13.1

6

92.8 ± 17.3

6

68.8 ± 31.9

6

0.0033

92.9 ± 9.0

6

79.0 ± 14.4

6

42.3 ± 14.2

6

0.01

101.4 ± 25.5

6

65.6 ± 25.6

6

31.5 ± 11.5

6

0.033

73.5 ± 34.3

6

33.1 ± 14.5

6

13.6 ± 15.6

6

0.33

66.9 ± 62.9

5

1.5 ± 3.6

6

14.6 ± 6.5

6

1.0

119.9 ± 6.7

3

0.0 ± 0.0

3

0.0 ± 9.2

3

33

18.7 ± 43.3

3

0.0 ± 0.0

3

0.0 ± 11.6

3

 

IC50cyt= 500µM

 

IC50diff= 0.024µM

 

 

 

NQ = non-quantifiable due to lack of inhibition of cell viability or differentiation

In experiment 1, 50% cytotoxicity was observed at the highest concentration, 1000µM (individual well results not reported), while less than 30% cytotoxicity was evident in all concentrations in experiment 2 (Table 2, average of 2 experiments). Therefore, it was not possible to calculate the average IC50cyt(50% inhibition of cell viability and growth).

It was not possible to apply the Foci Quantification method to determine the chondrocyte differentiation (IC50diff), as the number of foci was > 50% at all tested concentrations. Determining the chondrocyte differentiation using the Alcian Blue Elution method was shown to be inaccurate when comparing the data to the results of manual count of foci in the culture. Therefore these results were disregarded. For all the cytotoxicity measurements the positive control reduced cell viability/differentiation by > 80%, and was therefore valid.

Embryotoxic potential:

 

Table 2: Analysis of embryotoxic potential, 2-fold rule method

 

IC50diff(nM)

IC50cyt(nM)

IC50 ratio

Prediction

Run average

AMPD

1000000/NQ

1000000/NQ

1.0/na

Non-embryotoxic

atRA

24.25

500014

0.25

Embryotoxic

Run No. 1

AMPD

1000000

1000000

1.00

Non-embryotoxic

atRA

13.6

27.6

0.49

Embryotoxic

Run No. 2

AMPD

NQ

NQ

NQ

Non-embryotoxic

atRA

34.90

1000000

0.00003

Embryotoxic

NQ = non-quantifiable, an IC50 could not be calculated

 

In the 2 -fold rule method AMPD was predicted to be non-embryotoxic (see Table 2). The positive control was correctly predicted to be embryotoxic and therefore considered to be valid.

 

Table 3: analysis of embryotoxic potential, prediction model method

 

IC50diff(µg/mL)

Function I

Function II

Function III

Prediction

Run average

AMPD

105.1/NQ

3.95/NQ

4.16/NQ

-4.07/NQ

Non-embryotoxic

atRA

0.0073

-24.01

-21.74

1.44

Strongly embryotoxic

Run No. 1

AMPD

105.1

3.9537

4.1631

-4.0683

Non-embryotoxic

atRA

0.0041

-25.37

-23.0

1.71

Strongly embryotoxic

Run No. 2

AMPD

NQ

NQ

NQ

NQ

Non-embryotoxic

atRA

0.0105

-22.65

-20.48

1.17

Strongly embryotoxic

NQ = non-quantifiable, an IC50 could not be calculated

In the prediction model method AMPD was predicted to be non-embryotoxic (see Table 3). The positive control was correctly predicted to be strongly embryotoxic and therefore considered to be valid.

Applicant's summary and conclusion

Conclusions:
AMPD was predicted to be non-embryotoxic in the rat embryo limb bud micromass assay.
Executive summary:

The purpose of this study was to evaluate the embryotoxicity potential of 2-amino-2-methyl-1,3-propanediol (AMPD) in rats using the limb bud micromass assay. This preliminary screen involves high density culture of undifferentiated mesenchymal cells isolated from gestation day (GD) 13 rat embryo limb buds whereby during the 96 hour culture the cells proliferate, condense and differentiate into chondrocytes. Limb bud micromass cultures were exposed to six different concentrations of each test material. At the end of the culture period, the cells were assessed to determine the concentration at which 50% of the vehicle control group cell viability (IC50cyt) or chondrocyte differentiation (IC50diff) was inhibited. A neutral red updake assay was used for the cell viability/cytotoxicity evaluation. Inhibition of chondrocyte differentiation was assessed by staining the chondrocytes with alcian blue followed by 1) manually counting the stained foci, and 2) measurement of the alcian blue absorbance following elution from the foci. The embrotoxicity potential of AMPD was analyzed using two established methods (2-fold rule, prediction model) which assess the relative sensitivity for effects on differentiation versus cytotoxicity. Two independent trials were conducted for the test materials and a positive control inhibitor of chondrocyte differentiation, all-trans retinoic acid (atRA). Cell viability was marginally reduced following AMPD treatment (IC50cyt = 1000 μM). Inhibition of chondrocyte differentiation was marginally reduced following AMPD treatment. Due to limited or no inhibition of chondrocyte differentiation an IC50diff value could not be quantified for AMPD. Using the 2-fold rule and prediction model methods, AMPD was predicted to be non-embryotoxic. The known human and rat teratogen, atRA, was correctly predicted to be embryotoxic or strongly embryotoxic using the 2-fold rule or prediction model method, respectively.