Fatty acids, sunflower-oil, conjugated, maleated,reation products with diethanolamine, maleated tall-oil fatty acids and triethanolamine

Specific investigations: other studies

Administrative data

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-documented mechanistic investigation which meets basic scientific principles

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Diethanolamine Induces Hepatic Choline Deficiency in Mice

Author:

Lehman-McKeeman, L.D. et al.

Year:

2002

Bibliographic source:

Toxicological sciences 67, 39-45 (2002)

Reference 2

Reference Type:

publication

Title:

TOXICOLOGICAL HIGHLIGHT Choline Deficiency Associated with Diethanolamine Carcinogenicity

Author:

Newberne, P.M.

Year:

2002

Bibliographic source:

Toxicological Sciences 67, 1-3

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A subacute screening study on effects of dermally administered DEA on choline (Cho) and metabolites (phosphocholine (PCho); glycerophosphocholine (GCP); phosphatidylcholine (PC); s-adenosylmethionine (SAM); s-adenosylhomocysteine (SAH) was examined in groups of 6 – 8 male mice of two different strains.

GLP compliance:

not specified

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-iminodiethanol

EC Number:

203-868-0

EC Name:

2,2'-iminodiethanol

Cas Number:

111-42-2

Molecular formula:

C4H11NO2

IUPAC Name:

2,2'-iminodiethanol

Details on test material:

DEA (99% purity) was obtained ftom Aldrich Chemical Co. (Milwaukee, WI) . Absolute ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KY) was used to prepare 95% ethanol used as the vehicle in these studies. Betaine (HCl salt) was obtained from Aldrich, SAM and SAH were obtained from Sigma Chemical Co. (St. Louis, MO), and all standards for choline analyses were as described by Fornfict el al. (1989) .

Test animals

Species:

mouse

Strain:

other: B6C3F1 and C57BL6

Sex:

male

Details on test animals or test system and environmental conditions:

Male B6C3F1 mice, approximately 5 weeks of age at receipt, mice were housed in a temperature- and humidity-controlled environment and acclimated for approximately 1 week prior to study initiation. Unless otherwise noted, were allowed rodent chow (Purina 5001 ; Ralston-Purina. St. Louis, MO) and water ad libitum throughout the study. Dietary concentrations of choline and methionine in the Purina diet were 0.2 and 0.4%, respectively, whereas the fat composition was approximately 5%.
The same holds true for the male C57BL/6 mice.

DIETARY CHOLINE DEFICIENCY
Male B6C3F1 and C57BL6 mice (approximately 6-weeks old at study initiadon) were allowed ad libitum exposure to a control or choline-devoid diet (Dyets, Bethlehem, PA) for a period of 2 weeks (n = 8/group). The control diet contained 0.25% choline (as choline bitartrate) and the methionine content in the control and choline-devoid diets was 0.6% . At the end of the 2-week exposure period, livers were rapidly removed under anesthesia, immediately flash-frozen in liquid N, and stored at -80°C pending analysis of choline metabolites. In a separate group of mice (n - 4/group), blood was collected for clinical chemistry analyses, and livers were weighed and fixed in 10% neutral buffered formalin for histopathological assessment.

Administration / exposure

Route of administration:

dermal

Vehicle:

ethanol

Details on exposure:

For dosing, the backs of all mice were shaved, and the dosing solutions (1.8 ml/kg) were applied to a region approximately 2 cm2 (from the mid-back to the interscapular region) using a pipette . All mice, including the untreated controls, were shaved on an as-needed basis during the study period . At the end of the dosing or recovery period, livers were harvested as described above and analyzed for choline metabolites.
To evaluate potential strain differences in the response to DEA, male C57BL/6 mice, also about 6-weeks-old at study initiation, were dosed with 0 or 160 mg, DEA/kg for a similar 4-week period. The untreated and ethanol-treated control groups were also used in this study.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 20, 40, 80 and 160 mg/kg
Basis:

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

Male B6C3F1 mice (approximately 6-weeks old at study initiation) were housed individually and dosed dermally with DEA dissolved in 95% ethanol at 0, 10, 20, 40, 80, or 160 mg/kg (n = 6/group) for a period of 4 weeks (5 days/week). These dosages were selected to include those shown to be carcinogenic (40, 80, and 160 mg/kg/day; NTP, 1999). There were 2 control groups for this study including untreated and ethanol treated mice. A separate group of mice was dosed with the ethanol vehicle or 160 mg DEA/kg/day for 4 weeks (5 days/week) and then allowed a 2-week recovery prior to necropsy . For dosing, the backs of all mice were shaved, and the dosing solutions (1.8 ml/kg) were applied to a region approximately 2 cm2 (from the mid-back to the interscapular region) using a pipette . All mice, including the untreated controls, were shaved on an as-needed basis during the study period . At the end of the dosing or recovery period, livers were harvested as described above and analyzed for choline metabolites.
To evaluate potential strain differences in the response to DEA, male C57BL/6 mice, also about 6-weeks-old at study initiation, were dosed with 0 or 160 mg, DEA/kg for a similar 4 -week period. The untreated and ethanol-treated control groups were also used in this study.

Examinations

Examinations:

Analysis of hepatic choline metabolites

Results and discussion

Details on results:

After dermal treatment of B6C3F1 mice with DEA at 0, 10, 20, 40, 80, 160 mg/kg bw/day for 4 weeks, PCho was most sensitive to DEA treatment, decreasing at ≥20 mg/kg bw/day. GPC, choline, and PC also decreased in a dose-dependent manner. At ≥80 mg/kg bw/day, SAM levels decreased, while SAH levels increased in liver. The no-observed effect level (NOEL) for DEA induced changes was 10 mg/kg bw/day. Choline metabolites, SAM and SAH returned to control levels in mice allowed a 2-week recovery period. No fatty change was observed in the liver of DEA treated mice in a manner similar to dietary choline deficiency. In C57BL/6 mice, DEA treatment also decreased PCho concentrations, without affecting hepatic SAM levels. Dermal application of 95% ethanol for 4 weeks alone decreased hepatic betaine levels, suggesting that the use of ethanol as a vehicle for dermal application of DEA may exacerbate or confound the biochemical actions of DEA.

Any other information on results incl. tables

Effect of dietary choline deficiency on hepatic choline metabolites

Parameter	Control	Choline-devoid
Body weight (g)	24.8±0.4	24.9±0.6
Liver/BW (%)	5.1±0.1	4.8±0.1
PCho (nmol/g liver)	1146±100	297±53
GPC (nmol/g liver)	615±83	460±27
Cho (nmol/g liver)	139±4	95±18
PC (umol/g liver)	16.6±1.6	15.2±0.6
SAM (nmol/g liver)	69.2±4.2	56.8±3.3
SAH (nmol/g liver)	31.8±2.3	41.1±2.4

Effect of DEA Treatment on Hepatic Choline Metabolites

	DEA (mg/kg/day)
Parameter	0	10	20	40	80	160	160-R
Body weight (g)	26.6±0.8	25.9±0.4	26.6±0.1	25.8±0.4	26.1±0.4	27.1±0.6	25.8±0.8
Liver/BW (%)	5.6±0.1	5.6±0.1	5.7±0.1	5.5±0.1	5.5±0.1	6.2±0.1	5.5±0.1
PCho (nmol/g liver)	1220±44	1192±77	994±78	959±70	831±52	615±37	1224±114
GPC (nmol/g liver)	372±34	463±34	303±32	275±14	281±23	193±20	318±38
Cho (nmol/g liver)	155±14	137±12	152±11	126±7	94±14	106±13	180±21
PC (umol/g liver)	19.6±1.0	17.7±1.0	17.0±0.4	18.0±1.0	17.4±0.8	16.8±0.4	17.7±0.7
SAM (nmol/g liver)	83.9±1.1	84.1±4.1	84.7±3.9	82.1±7.2	65.1±6.4	53.3±4.3	84.9±1.0
SAH (nmol/g liver)	48.1±1.9	52.2±6.2	49.1±1.8	52.6±1.4	61.8±4.4	58.5±2.7	46.1±4.2