Methyl 12-hydroxyoctadecanoate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The analogue approach is used for the hazard assessment of toxicological, eco-toxicological and environmental fate endpoints for the registration of 12-hydroxstearate methyl ester (CAS 141-23- 1). The hypothesis is that data can be read-across between this ester and its structural analogues, based on structural similarity and which cause the same type of effect(s) in physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015). The primary fatty acids in this read-across are lauric acid (C12) and myristic acid (C14), as these are well studied with high-quality experimental data. Supplemental analogues are used which contribute understanding of the effects of other structural features not contained in the two primary analogues.

There are no GHS classifications for 12-hydroxstearate methyl ester for endpoints which are reliant on read-across. There is a high degree of confidence that hazards for these endpoints are not underestimated, based on a strong weight of evidence from multiple data sources.

Read-across data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is used for fulfilling the data requirements of the REACH registration and classifying potential hazards. This read-across approach is adequate for the purposes of risk assessment and classification and labeling.

Reason / purpose for cross-reference:

read-across source

Objective of study:

absorption

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Hydroxylated fatty acids were assayed in the fat of rats fed up to 10% hydrogenated castor oi In a subchronic dietary study

GLP compliance:

not specified

Specific details on test material used for the study:

Hydrogenated Castor Oil (HCO) was a granulated powder containing 86.5% 12-hydroxystearic acid, 10.3% nonoxygenated acids, and 3.2% 12-ketostearic acid. The melting point was reported to be above 80 degrees C.

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Slonaker substrain of albino Wistar strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: male and female rats (Slonaker substrain of albino Wistar strain). The preliminary studies utilized three weanling female rats per dose group; the main study utilized 15 male rats per dose group (body weight ranging from 43 to 83 g). They were maintained on Purina Laboratory Chow. Food and water were available ad libitum. Body weight and food consumption records (by group) were maintained weekly. Live weight data were statistically analyzed at the end of the experiment to indicate the influence of diet. Differences in initial weight were removed in order to compare live weights on the basis of the same initial weight for each rat. Weights of all rats living at a particular time were included in calculations of average weight and weight gain.

Route of administration:

oral: feed

Vehicle:

corn oil

Dose / conc.:

0 other: %

Dose / conc.:

1 other: %

Remarks:

(w/w), dissolved in corn oil

Dose / conc.:

10 other: %

Remarks:

(w/w), dissolved in corn oil

No. of animals per sex per dose / concentration:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Diets were prepared from Purina Laboratory Chow. For the first preliminary study, HCO in the form of a powder was added to the chow at the expense of an equal amount of the rat diet. For the second preliminary and main studies, HCO was melted and mixed with corn oil, which was then blended with the chow (representing 80% of each diet).

Details on dosing and sampling:

In the main study, fifteen male rats were assigned to each dose group in the main study in order to give the same mean weight per group. Animals were fed the diet for eight weeks, then half of the rats on the HCO diets were given the control corn oil diet until the end of the experiment at 16 weeks. Data was generated for the following 4 groups: 1% HCO for 16 weeks; 1% HCO for 8 weeks, then corn oil diet for 8 weeks; 10% HCO for 16 weeks; 10% HCO for 8 weeks, then corn oil diet for 8 weeks. Body weights were statistically analyzed (p ≥ 0.05) to indicate the influence of diet.

After 4 weeks of feeding, some of the animals on the 1% HCO (three rats) and 10% HCO (three rats) diets were necropsied and excised abdominal adipose tissue from each group was pooled. At weeks 4 and 8, sets of three rats on the corn oil diet were used. At the end of the 16-week feeding period, adipose tissue from rats in the various dietary groups was analyzed. Adipose tissue was not pooled. After 8, 12, and 16 weeks, lipids were extracted from three rats on each diet. Methyl esters were recovered from the extracted lipids and chromatographed. Extractions of duplicate carcass fat samples were reproducible within 4-5%.

Statistics:

Group differences were considered significant at p< 0.05. Reproducibility was greater than 95%.

Type:

absorption

Results:

Hydroxylated fatty acids were detected in carcass and adipose fat of rats fed hydrogenated castor oil in the diet.

Details on absorption:

Hydroxystearic acid was deposited in abdominal fat and other body lipids. In all cases, it was accompanied by hydroxypalmitic acid, hydroxymyristic acid, and hydroxylauric acid (all are hydroxy-stearic acid metabolites). The percent composition of these HCO-derived hydroxy fatty acids in rat lipids was as follows: 12-hydroxystearic acid (81%), 10-hydroxypalmitic acid (17%), 8-hydroxymyristic acid (1.6%), and 6-hydroxylauric acid (0.4%). This is consistent with metabolism which occurs by successive losses of two-carbon units from the carboxyl end of the fatty acid chain.
Hydroxylipids increased in the animals fed both concentrations of HCO in the diet, as a percentage of dry carcass weight. After 4 weeks into the main experiment in animals on the 1% HCO diet, HCO-derived fatty acids accounted for 0.90% (by weight) of the abdominal fat fatty acids. The maximum content of HCO-derived hydroxyacids in body lipids was 4.4% in abdominal fat at 4 weeks on the 10% HCO diet. It appears that hydroxyacids derived from HCO in the diet preferentially find their way into abdominal fat compared to other body (carcass) lipids. Compared to the abdominal fatty acids, the acids obtained from the carcass lipids contained a smaller proportion (0.28% over the 8- to 16-week feeding period) of HCO-derived hydroxyacids. The total content of HCO-derived hydroxyacids (combined abdominal fat and carcass lipids) was 6.28% in rats fed 10% HCO in the diet for 8 weeks.

When rats were changed from the HCO diet to the control diet, a rapid decrease in the amount of HCO-derived hydroxy fatty acids in the tissues occurred. Loss of hydroxyl fatty acids was rats on the 10% HCO diet was more pronounced that in those on the 1% HCO diet. The proportion of HCO-derived hydroxyacids decreased from 0.90% to 0.35% over the 8- to 16-week feeding period in animals fed the 1% HCO diet. In animals fed 10% HCO in the diet for only 8 of 16 weeks, this proportion had decreased from 6% to <2%, equally distributed in abdominal fat and carcass lipids. Hydroxystearic acid was also found in animals on the control diet (corn oil), consistent with other studies examining the content of hydroxyacids in the rat body, as well as in humans and canines, and suggests that hydroxyacid formation is a normal phenomenon.

Details on distribution in tissues:

Hydroxyacids were found in adipose tissue and carcass fat after the feeding of rats of up to 10% HCO in the diet. This is consistent with lipid formation from fats and triglycerides in the gut after intake of lipid containing food.

Details on excretion:

no data

Metabolites identified:

yes

Details on metabolites:

Hydroxystearic acid was deposited in abdominal fat and other body lipids. In all cases, it was accompanied by hydroxypalmitic acid, hydroxymyristic acid, and hydroxylauric acid (all are hydroxy-stearic acid metabolites). The percent composition of these HCO-derived hydroxy fatty acids in rat lipids was as follows: 12-hydroxystearic acid (81%), 10-hydroxypalmitic acid (17%), 8-hydroxymyristic acid (1.6%), and 6-hydroxylauric acid (0.4%). This is consistent with metabolism which occurs by successive losses of two-carbon units from the carboxyl end of the fatty acid chain.

Table 1: Mean Hydroxy Fatty Acid Content in Rats (Binder et al., 1970).

 

		Week
Regimen	Tissue	4	8	12	16
		Weight Percent of carcass fatty acids
Corn Oil	Abdom fat	-	-	0.027	0.020
	Carcass	-	0.43	0.022	0.023
1% HCO	Abdom fat	0.97	0.34	0.36	0.35
	Carcass	-	0.27	0.29	0.28
1% HCO x 8 wks then corn oil x 8 wks	Abdom fat	-	-	0.087	0.048
	Carcass	-	-	0.079	0.042
10% HCO	Abdom fat	4.40	4.03	2.28	1.67
	Carcass	-	2.25	1.81	1.91
10% HCO x 8 wks then corn oil x 8 weks	Abdom fat	-	-	0.87	0.12
	Carcass	-	-	0.27	0.10

Standard deviation values are available in the original reference article

 

Conclusions:

Rats ingesting 1% and 10% hydrogenated castor oil (HCO) dissolved in corn oil (0.865% or 8.65% equivalent) in a laboratory chow diet for 8-16 weeks showed increased accumulation of hydroxy fatty acids in body fat reserves (primarily adipose tissue) when compared with rats on a control diet (20% corn oil). This is consistent with utilization of hydroxylated fatty acids (from HCO) in the synthesis of triglycerides stored in fat tissue. Hydroxy fatty acids were present at a low level in the fat of rats fed the control diet

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The analogue approach is used for the hazard assessment of toxicological, eco-toxicological and environmental fate endpoints for the registration of 12-hydroxstearate methyl ester (CAS 141-23- 1). The hypothesis is that data can be read-across between this ester and its structural analogues, based on structural similarity and which cause the same type of effect(s) in physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015). The primary fatty acids in this read-across are lauric acid (C12) and myristic acid (C14), as these are well studied with high-quality experimental data. Supplemental analogues are used which contribute understanding of the effects of other structural features not contained in the two primary analogues.

There are no GHS classifications for 12-hydroxstearate methyl ester for endpoints which are reliant on read-across. There is a high degree of confidence that hazards for these endpoints are not underestimated, based on a strong weight of evidence from multiple data sources.

Read-across data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is used for fulfilling the data requirements of the REACH registration and classifying potential hazards. This read-across approach is adequate for the purposes of risk assessment and classification and labeling.

Reason / purpose for cross-reference:

read-across source

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Specific details on test material used for the study:

Purity of ethyl oleaste was 99.0%; triolean was also used as a control and was 98.7%.

Radiolabelling:

yes

Remarks:

Specific activity was 165 µCi/mg . Radiolabel was on the carboxy carbon.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sprague- Dawley rats [Crl:C D (SD)IGS BR] were obtained from Charles River Laboratory. At dosing, the animals weighed 194-253 g and were approximately 8-10 weeks of age. Rats were housed individually in glass metabolism cages designed for the separation and collection of urine, feces, and expired air. Temperature and humidity were controlled throughout the duration of the study. A certified standard rodent diet (Harlan Teklad) and water were available, ad libitum, at all times throughout the study period. Mortality and moribundity checks were done twice daily and cage-side observation for general health and appearance was done once daily.

Route of administration:

oral: gavage

Vehicle:

other: Triglycerol mono-oleate (Caprol 3GO) and polysorbate 80 (Tween 80)

Remarks:

2 and 1%, w/w, respectively

Details on exposure:

Formulated as a 34% w/w lipid emulsion for dosing (non-radiolabeled ethyl oleate was used as the carrier for the labeled ethyl oleate, and high oleic safflower oil was us the carrier for the labeled triolein (control). Triglycerol mono-oleate (Caprol 3GO) and polysorbate 80 (Tween 80) were used as emulsifiers (2 and 1%, w/w, respectively).

Specific activityof the radiolabeled ethyl oleate was 165 µCi/mg, and of the triolein, 117.5 µCi/mg. The radiolabel was on the carboxyl carbon. HPLC analysis showed the mean radiopurity of [14C]ethyl oleate and [14C]triolein to be 99.0 and 98.7%, respectively

Duration and frequency of treatment / exposure:

One oral dose

Dose / conc.:

1.7 other: g/kg body weight

Dose / conc.:

3.4 other: g/kg body weight

No. of animals per sex per dose / concentration:

5

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

Urine and feces were collected every 24 h until 72 h post dose. Expired air was paased through an activated charcoal trap to recover volatile organic compounds, then carbon dioxide trapping solution to collect radiolabeled carbon dioxide. Expired air was measured at 6,12, 18 and 24 h post-dose, and then at 24 h intervals thereafter until 72 h post-dose. Animals were sacrificed at 72 h post-dose by exsanguination (cardiac puncture) under halothane anesthesia and tissues, including blood, were collected for analysis of radioactivity in each matrix. All samples were homogenized or mixed prior to radioanalysis, which was performed by either direct liquid scintillation counting (LSC) or by combusion followed by LSC. All samples were analyzed in duplicate, and analysis was repeated, sample size allowing, if variability between replicates was more than 10%.

Statistics:

Statistical analyses were limited to simple expressions of variation, including mean and standard deviation.

Type:

absorption

Results:

70-90% of the EO test material

Type:

distribution

Results:

At sacrifice, the highest radioactivity: 84-89% of tissue radioactivity was found in the residual carcass (after removal of internal organs). The tissue with highest radioactivity was mesenteric fat (0.4-0.7% for EO and 0.4-1.8% for TG.)

Type:

excretion

Results:

Primarily via expired air (40-70% administered dose); feces. No significant excretion in urine

Details on absorption:

Both doses of test material were well absorbed with approximately 70-90% absorbed. Approximately 90-100% of the TG dose was absorbed.

Details on distribution in tissues:

At 72 h post administration, tissue distribution of the test material and the TG control was similar. At sacrifice, the vast majority of radioactivity was found in the residual carcass (84-89% of total tissue radioactivity), which is left after all internal tissues have been removed, and primarily consists of muscle skin and fat). This is likely the result of this being the greatest mass and weight. The specific soft tissue with the highest concentration of radioactivity in both groups and sexes was mesenteric fat. The other organs and tissues had very low concentrations of test-material derived radioactivity.

Key result

Test no.:

#1

Transfer type:

other: distribution into mesenteric fat

Remarks:

Low dose 1.7 g/kg bw

Observation:

distinct transfer

Remarks:

normal distribution of lipid

Key result

Test no.:

#2

Transfer type:

other: distribution into mesenteric fat

Remarks:

High dose 3.4 g/kg bw

Observation:

distinct transfer

Remarks:

normal distribution of lipid

Key result

Test no.:

#3

Transfer type:

other: distribution into mesenteric fat

Remarks:

control TG (triolein)

Observation:

distinct transfer

Remarks:

normal distribution of lipid

Details on excretion:

Both doses of test material were rapidly and extensively excreted as CO2 with no remarkable differences between their excretion profiles. Approximately 40-70 % of the administered dose for both groups was excreted as CO2 within the first 12 h. A secondary route of elimination is via feces for all groups. Fecal elimination of ethyl oleate (EO) appeared to be dose dependent, with 7-8% of the low dose and 20% of the high dose excreted in the feces. Urinary excretion was minimal for both compounds.

Metabolites identified:

yes

Remarks:

CO2

Details on metabolites:

Primary metabolite is CO2 exhaled, supporting the known metabolic pathway of β-oxidation of lipids with liberation of CO2.

Bioaccessibility (or Bioavailability) testing results:

High absorption suggests high bioaccessibility.

All animals appeared healthy and showed no signs of toxicity throughout the study. Feces of all animals appeared normal.

Overall mean total recovery (mass balance) of radioactive dose by 72 h ranged from 93 -102% for all treatment groups.

Conclusions:

A pharmacokinetic study in rats examined the absorption, distribution and excretion of orally-administered radiolabeled ethyl oleate and a control triacylglycerol (TG, triolein) lipid. The ethyl oleate, at 2 doses, was highly absorbed (70-90%), as was the control triolein (TG). The primary route of excretion was via exhaled CO2 (40-70% of administered dose), and secondarily, feces. No significant urinary excretion was noted. The main repository into which the ethyl oleate radiolabel was deposited was residual carcass, while the specific soft tissue mesenteric fat; these were similar to that for the control TG. These data provide evidence that the oleic acid moiety is utilized in the body as endogenous TG-derived fatty acids.

Description of key information

A structural analogue and a metabolic precursor prove to be highly absorbed after orally administered and metabolised. Dermal absorption is minimal due to low water solubility and high log Kow (6.69). The substance is not volatile.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

90

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

10

Additional information

The results of a pharmacokinetic study in rats demonstrate that the absorption, distribution and excretion of orally-administered radiolabeled ethyl oleate (EO) is rapid and approximately 80% of the administered high dose (10% in the diet) (Bookstaff, et al., 2004). This was reported earlier by experiments of ADME of EO. Absorption of EO may be slightly lower than that of TG. Esters of fatty acids are rapidly hydrolyzed to ethanol and free fatty acids within the GI tracts (Froyland, et all, 1996). Saghir, et al, 1997, demonstrated that fatty acid ethyl esters are degraded to free fatty acids in the duodenum with a short half-life. This is supported in the current study by the observation of rapid exhalation of radioactive CO2, as oxidation of fatty acids requires a free carboxyl end (i.e., not esterified). The rate and extent of radiolabeled CO2 excretion was similar between the ethyl oleate and the TG groups, indicating that both types of lipid deliver the free fatty acid to tissues for oxidation at similar rates. This provides evidence that the oleic acid moiety is utilized in the body in a manner similar to that of an endogenous TG-derived fatty acid.

In a supporting study by Binder, et al. from 1970, prior to the establishment of an OECD guideline method for a toxicokinetic study, hydroxyacids and metabolites were documented in body fat in a dose- and time-related manner.  Rats were fed diets high in either 1% or 10% hydrogenated castor oil (HCO, a triglyceride of 12-hydroxystearic acid) for 8 or 16 weeks, and hydroxylated lipids were found to be deposited into abdominal fat and carcass fat. The lipid was biochemically identified to be hydroxystearic acid (C-18) along with hydroxypalmitic acid (C-16), hydroxymyristic acid (C-14), and hydroxylauric acid (C-12). The percent composition of these HCO-derived hydroxy fatty acids in rat lipids was as follows: 12-hydroxystearic acid (81%), 10-hydroxypalmitic acid (17%), 8-hydroxymyristic acid (1.6%), and 6-hydroxylauric acid (0.4%). 

The amount of the hydroxy lipid in body fat was dose-related, as rats fed the 1% HCO diet showed 0.90% hydroxy lipid (by weight) within the total abdominal fat fatty acids, whereas the maximum content of HCO-derived hydroxyacids was 4.4% in abdominal fat in animals on the 10 % HCO diet.  In groups of animals switched from the diet of hydrogenated castor oil after 8 weeks to one of corn oil for an additional 8 weeks, the percentage of the hydroxy fatty acids in rat lipids rapidly dropped (from 0.90% to 0.35% in animals fed the 1% HCO diet, and from 6% to <2% in those fed the 10% HCO diet.)

These data support the hypothesis that dietary 12-hydroxystearic acid enters the normal lipid metabolic pathways of the gut based on lipolytic cleavage, absorption through the wall of the small intestine and ultimate catabolism by the process β-oxidation. This is the metabolic process which occurs by successive losses of two-carbon units from the carboxyl end of the fatty acid chain.