Dimethylamine

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no guideline followed, well documented experimental result

Objective of study:

absorption

excretion

other: secretion

Principles of method if other than guideline:

no guideline followed, details on method are given below

GLP compliance:

no

Radiolabelling:

not specified

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Male 15- to 20-week-old Wistar rats were fed a commercial diet containing 23.6 mg/kg DMA or a low-DMA diet containing 1.0 mg/kg DMA. The breeding room was maintained at 25 C and 60% humidity. The components of the low-DMA diet were 68% corn starch, 20% soy casein, 5% soya bean oil, 2% cellulose, 4% minerals and 1% vitamin mixture.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The low-DMA diet or the commercial diet and tap water were given freely to rats for one week prior to sacrifice.

Duration and frequency of treatment / exposure:

details are given below

Remarks:

Doses / Concentrations:
details are given below

No. of animals per sex per dose / concentration:

details are given below

Control animals:

not specified

Positive control reference chemical:

details are given below

Details on study design:

details are given below

Details on dosing and sampling:

Rats were anaesthetized by intraperitoneal injection of sodium pentobarbital, and the stomach, the small intestine, the caecum and the large intestine were separated. The small intestine was cut into four equal lengths. The intestinal contents were washed out with 10 mL 0.05 mol/L hydrochloric acid. Blood was obtained by cardiac puncture. Killing was carried out in the morning.

Absorption, secretion and excretion tests
Rats fed the commercial diet were fasted overnight and anaesthetized. The abdomen was opened and the stomach and the caecum were ligated at both ends without closing the blood vessels. The upper and lower small intestine and the large intestine were separated into sections of about 5 cm in length by ligation. The site of the ligated section in the upper small intestine was 10 cm from the pylorus and that in the lower small intestine 10 cm from the ileo-caecal valve; 250 µg DMA were injected into each ligated section for the absorption tests. After a given time, the ligated sections were removed and the intestinal contents were washed out with 10 mL 0.05 mol/L hydrochloric acid. For the secretion test, an equal dose of DMA was injected through a femoral vein, and the ligated upper small intestine was removed at a given time. Urine in the bladder was collected through a syringe.

Statistics:

details are given below

Preliminary studies:

details are given below

Details on absorption:

All rates of disappearance of DMA from the stomach and the intestines after injection of 250 µg DMA were monoexponential over 30 min. The biological half-lives were 8.3, 11.6, 31.5 and 11.0 min for the upper small intestine, the lower small intestine, the caecum and the large intestine, respectively. Absorption of DMA from the stomach was barely observable (t1/2 = 198 min).

DMA was not absorbed from the stomach, but was absorbed from the small intestine, the caecum and the large intestine.

Details on distribution in tissues:

Distribution of DMA in the digestive tract
DMA in the contents of the gastrointestinal tract and in blood was determined after the rats had been fed the commercial or the low-DMA diet for a week. The concentration of DMA in the gastrointestinal tract of the rats fed the commercial diet was high in the upper intestine and low in the lower intestine. When rats were fed the low-DMA diet, the DMA concentration was lowest (1.3 ± 0.5 mg/kg) in the stomach and highest (6.6 + 2.5 mg/kg) in the upper smalt intestine and decreased in the lower small intestine and the caecum. The DMA concentration in the large intestine was higher than that in the caecum in both groups. The concentration of DMA in the contents of each ligated section in the rats fed the low-DMA diet was lower than that of the corresponding section in rats fed the commercial diet. Less than 1 mg/kg DMA was found in blood in both groups. The DMA concentration in the intestinal contents of rats fed the low-DMA diet was higher than that in the diet, but that in the intestinal contents of the rats fed the commercial diet was lower than that in the diet.

Details on excretion:

Decrease of DM A levels in blood and intestinal secretion
The initial half-life of DMA in blood was 12.5 min, and the disappearance curve was monoexponential. Urinary DMA concentration increased from 17.3 ± 9.4 mg/kg to 139 + 23 mg/kg in 30 min. DMA was excreted not only in urine but also in the small intestine. The highest concentration of intestinal DMA (15.6 ± 12.6 mg/kg) was observed 15 min after intravenous injection of DMA. When the intestinal DMA level decreased to the basic concentration, the blood DMA increased a little. The half-life for secondary disappearance of DMA in blood was 15.2 min.

Most of the DMA in blood was excreted in the urine in a relatively short time and a small portion was secreted in the intestine.

Details on metabolites:

details are given below

Distribution of DMA in the digestive tract

DMA in the contents of the gastrointestinal tract and in blood was determined after the rats had been fed the commercial or the low-DMA diet for a week. The concentration of DMA in the gastrointestinal tract of the rats fed the commercial diet was high in the upper intestine and low in the lower intestine. When rats were fed the low-DMA diet, the DMA concentration was lowest (1.3 ± 0.5 mg/kg) in the stomach and highest (6.6 + 2.5 mg/kg) in the upper smalt intestine and decreased in the lower small intestine and the caecum. The DMA concentration in the large intestine was higher than that in the caecum in both groups. The concentration of DMA in the contents of each ligated section in the rats fed the low-DMA diet was lower than that of the corresponding section in rats fed the commercial diet. Less than 1 mg/kg DMA was found in blood in both groups. The DMA concentration in the intestinal contents of rats fed the low-DMA diet was higher than that in the diet, but that in the intestinal contents of the rats fed the commercial diet was lower than that in the diet.

Gastrointestinal absorption

All rates of disappearance of DMA from the stomach and the intestines after injection of 250 µg DMA were monoexponential over 30 min. The biological half-lives were 8.3, 11.6, 31.5 and 11.0 min for the upper small intestine, the lower small intestine, the caecum and the large intestine, respectively. Absorption of DMA from the stomach was barely observable (t1/2 = 198 min).

Decrease of DM A levels in blood and intestinal secretion

The initial half-life of DMA in blood was 12.5 min, and the disappearance curve was monoexponential. Urinary DMA concentration increased from 17.3 ± 9.4 mg/kg to 139 + 23 mg/kg in 30 min. DMA was excreted not only in urine but also in the small intestine. The highest concentration of intestinal DMA (15.6 ± 12.6 mg/kg) was observed 15 min after intravenous injection of DMA. When the intestinal DMA level decreased to the basic concentration, the blood DMA increased a little. The half-life for secondary disappearance of DMA in blood was 15.2 min.

Conclusions:

Interpretation of results: low bioaccumulation potential based on study results
After ingestion of DMA an intestinal absorption occurs, follwed by an accumulation into the blood. From here two pathways can be found: excretion via the urine (1; DMA leaved the body), or an intestinal secretion is performed, whereby this is followed one more time by an intestinal absorpition and an accumulation in the blood.

Executive summary:

DMA was not absorbed from the stomach, but was absorbed from the small intestine, the caecum and the large intestine. Absorbed DMA appeared in the blood, then disappeared rapidly. Most of the DMA in blood was excreted in the urine in a relatively short time and a small portion was secreted in the intestine. When rats were fed a commercial diet containing 23.6 mg/kg DMA, the concentration in the gastrointestinal tract was highest in the stomach and decreased from the upper intestine to the lower region. Since the DMA concentration in the lower intestines (about 4 mg/kg) was not affected by ingestion of DMA, most of the DMA ingested with the diet may be absorbed in the small intestine. The highest rate of absorption of DMA was observed in the upper small intestine, and was almost the same as that observed in guinea-pigs (Ishiwata et al., 1977) The DMA found in the caecum and the large intestine may include an endogenous contribution, as reported by Asatoor and Simenhoff (1965) and by Johnson (1977). These authors concluded that intestinal bacteria form DMA from lecithin. The gastrointestinal distribution of DMA in rats fed the low-DMA diet differed considerably from that in rats fed the commercial diet. The highest concentration of DMA (6.6 mg/kg), observed in the upper small intestine of rats fed the low-DMA diet, may be due to intestinal secretion of DMA from the blood. DMA secreted into the small intestine from blood can be re-absorbed. This is a probable explanation for the second maximum concentration of DMA observed in blood 25 min after intravenous injection of DMA. The disappearance rate between 25 and 30 min (t1/2 = 15.2) was almost the same as that between 5 and 15 min. The higher concentration of DMA in the large intestine than in the caecum is considered to be due to the absorption of intestinal moisture. The site of DMA formation can be considered to be the lower digestive tract, although it may not always be the site of the highest DMA concentration.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no guideline followed, well - described experimental result.

Objective of study:

excretion

Principles of method if other than guideline:

no guideline followed

GLP compliance:

no

Radiolabelling:

yes

Species:

other: rat and mouse (adult animals)

Strain:

other: Wistar and CD1

Sex:

male

Details on test animals or test system and environmental conditions:

adult male rat (68 ^Ci; 0-5 mL) (Wistar strain, 250 g; Harlan, Oxford Laboratory Animal Centre, Bicester, UK) and to adult male mouse (8 /*Ci; 0-1 mL) (CD1 strain; 25 g; Charles River, Manston, UK)
housing after drug-administration: The animals were then housed in separate glass metabolism cages ('Metabowls', Jencons Ltd, Hemel Hempstead, UK) for 3 days in an ambient environment with free access to food (' LabSure' CRM pellets; K.&K . Greef Ltd, Croydon, U K) and water being permitted 2 h after dosing.

Route of administration:

oral: gavage

Vehicle:

other: destilled water

Details on exposure:

administered at 20µmol/kg body weight by gavage, then an overnight fast.

Duration and frequency of treatment / exposure:

details are given below

Remarks:

Doses / Concentrations:
details are given below

No. of animals per sex per dose / concentration:

details are given below

Control animals:

other: details are given below

Positive control reference chemical:

details are given below

Details on study design:

details are given below

Details on dosing and sampling:

Urine and faeces were collected separately each day into flasks containing acid (HC11 M, 1 ml) with daily cage washings (diluted acid (HC1 100 mM) then water) being added to the relevant urine samples. Expired air was drawn through a series of Dreschel bottles containing ethanolamine/2-methoxyethanol (1/2 v/v) and examined after 3 days for trapped 14C02. This mixture, being alkaline, would not trap exhaled volatile amines. At the end of the study all animals were killed by cervical dislocation, frozen (- 20°C) and examined for retained radioactivity.

Statistics:

details are given below

Preliminary studies:

details are given below

Details on absorption:

dimethylamine is rapidly absorbed. The basic character of dimethylamine (pKa ~ 10-3) should favour its absorption from the more alkaline environments of the gastrointestinal tract and the intestine has been shown to be major site of absorption in rat, with negligible uptake occurring from the acidic environment of the stomach (Ishiwata et al 1984).

Details on distribution in tissues:

Cycling with secretion into the acidic environment of the stomach could take place.

Transfer type:

secretion via gastric mucosa

Observation:

distinct transfer

Details on excretion:

Virtually identical excretory profiles were obtained for rat and mouse following the oral administration of [14C]-dimethylamine (table 1). In both rodents, urine was the major route of excretion with the majority of radioactivity (91%) being voided during the first day. Additional small amounts of radioactivity were observed in the 24-72 h urine (2%), in faeces (2%) and in exhaled air (1%), with a further quantity (1%) being detected within the carcass after 3 days. Good overall recoveries were achieved, suggesting that only trace amounts of volatile compounds had been exhaled and escaped detection. The small amounts of radioactivity detected in the faeces suggests that this low-molecular weight compound is a poor candidate for biliary excretion, as expected and in alignment with previous findings where < 2% of a dose was found in bile (Ishiwata et al 1984).

Metabolites identified:

yes

Details on metabolites:

The majority of [14C]-dimethylamine administered was excreted unchanged in the urine with only small amounts being demethylated to [14C]-methylamine.
In all 0-24 h urine samples examined two radioactive areas were found, which corresponded to authentic methylamine and dimethylamine, but ideal resolution was not always achieved with tic (Zhang et al 1994). Reverse-phase paper chromatography of derivatized aliquots of urine revealed the presence of an area of radioactivity (i?f=0-56), which co-chromatographed with the 2,4-dinitrobenzene derivative of authentic dimethylamine. A separate minor radioactive area {R( = 0-64), which co-chromatographed with the corresponding derivative of authentic methylamine, was also present in derivatized aliquots of urine.

Metabolite identification and quantification

In all 0-24 h urine samples examined two radioactive areas were found, which corresponded to authentic methylamine and dimethylamine, but ideal resolution was not always achieved with tic (Zhang et al 1994). Reverse-phase paper chromatography of derivatized aliquots of urine revealed the presence of an area of radioactivity (i?f=0-56), which co-chromatographed with the 2,4-dinitrobenzene derivative of authentic dimethylamine. A separate minor radioactive area {R( = 0-64), which co-chromatographed with the corresponding derivative of authentic methylamine, was also present in derivatized aliquots of urine. These tentative assignments were confirmed by mass spectrometry (figure 1). Molecular ions were detected, as expected, at m\z 211 (65% abundance) and mjz 197 (85% abundance) corresponding to the 2,4-dinitrobenzene derivatives of dimethylamine and methylamine respectively. The base peak (100% abundance) for the methylamine derivative corresponded to a fragment ion at m\z 105, which had lost the two nitro groups. Although the corresponding fragment ion was observed for the dimethylamine derivative {mjz 119, 71% abundance), the base peak occurred at m\z 136 [M-75] + . Other fragment ions were assignable and in agreement with those obtained from synthetic standards and previously published spectra (Stenhagen et al 1974). Results obtained from the quantification of the two radioactive areas corresponding to the two derivatized amines within the first 0-24 h urine samples showed little variation between the species with 96-6 ± 2-5 and 95 5 ± 1-3% of the radioactivity being present as dimethylamine in rat and mouse respectively. The demethylation product, methylamine, only accounted for 3-4 ± 1-5 (rat) and 4-5 ± 1-3% (mouse) of the 0-24 h urinary radioactivity output.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Dimethylamine is rapidly absorbed and excreted via the urine.

Executive summary:

The present study suggests that dimethylamine is rapidly absorbed and excreted via the urine. The basic character of dimethylamine (pKa ~ 10-3) should favour its absorption from the more alkaline environments of the gastrointestinal tract and the intestine has been shown to be major site of absorption in rat, with negligible uptake occurring from the acidic environment of the stomach (Ishiwata et al 1984).

The small amounts of radioactivity detected in the faeces suggests that this low-molecular weight compound is a poor candidate for biliary excretion, as expected and in alignment with previous findings where < 2% of a dose was found in bile (Ishiwata et al 1984). However, cycling with secretion into the acidic environment of the stomach could take place. Indeed, the increased concentration of dimethylamine reported in the gastric fluid of dog, ferret and rat shortly after intravenous injection indicates secretion of the compound into the stomach (Ishiwata et al 1984, Zeisel et al 1986). The majority of [14C]-dimethylamine administered was excreted unchanged in the urine with only small amounts being demethylated to [14C]-methylamine. This demethylation pathway has been shown previously to occur in dog, ferret and rat (McNulty and Heck 1983, Zeisel et al 1985, 1986) with the majority (98%) of an inhaled dose of dimethylamine being extracted unchanged from the exposed rat (McNulty et al 1983). No metabolism has been detected in fish injected with [14C]-dimethylamine (Charest et al 1988).

The liberated methyl moiety is presumably oxidized and exhaled as [14C]-C02 or may be incorporated, perhaps via the intermediate [14C]-formaldehyde through the formyl (or formimino) group transfer activities of the one-carbon pool, into macromolecules that remain within the carcass. Previous studies where radiolabel was detected in the carcass of rat exposed to [14C]-dimethylamine vapour together with the observation of a long terminal half-life (55 h) in these animals (McNulty and Heck 1983) adds support to the later suggestion.

The liberated methyl moiety is presumably oxidized and exhaled as [14C]-C02 or may be incorporated, perhaps via the intermediate [14C]-formaldehyde through the formyl (or formimino) group transfer activities of the one-carbon pool, into macromolecules that remain within the carcass. Previous studies where radiolabel was detected in the carcass of rat exposed to [14C]-dimethylamine vapour together with the observation of a long terminal half-life (55 h) in these animals (McNulty and Heck 1983) adds support to the later suggestion.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable well-documented publication, which meets basic scientific principles

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Frosch PJ, Kligman AM: The soap chamber test: A new method for assessing the irritancy of soaps. J Am Acad Dermatol 1979;1:35–41.

Qualifier:

according to guideline

Guideline:

other: Pinnagoda J, Tupker RA, Agner T, Serup J: Guidelines for transepidermal water loss (TEWL) measurement: A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1990; 22:164–178.

Qualifier:

according to guideline

Guideline:

other: Rogiers V: EEMCO guidance for the assessment of transepidermal water loss in cosmetic sciences. Skin Pharmacol Appl Skin Physiol 2001;14:117–128

Qualifier:

according to guideline

Guideline:

other: Fullerton A, Fischer T, Lahti A, Wilhelm KP, Takiwaki H, Serup J: Guidelines for measurement of skin colour and erythema: A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1996;35:1–10

Qualifier:

according to guideline

Guideline:

other: Parra JL, Paye M: EEMCO Guidance for the in vivo assessment of skin surface pH. Skin Pharmacol Appl Skin Physiol 2003;16:188–202

GLP compliance:

no

Radiolabelling:

no

Species:

human

Strain:

other: not applicable

Sex:

male/female

Details on test animals or test system and environmental conditions:

The study was performed in a single-blinded, randomized manner under standardized laboratory conditions using air-conditioning with a room temperature between 20 and 22°C and a relative humidity between 30 and 40%.
20 healthy, non-preselected Caucasian volunteers (11 males and 9 females; aged 19–46 years, median age 28.3 years) without any skin or other systemic diseases were included. During the study period, the subjects were allowed to shower as usual, but they were instructed to avoid any application of detergents, emollients and moisturizers on their backs as well as natural or artificial UV exposure.

Type of coverage:

occlusive

Vehicle:

other: distilled water

Duration of exposure:

30 min, then 3 hours later again 30 min exposure, this treatment was performed on 4 consecutive days

Doses:

(0.01, 0.1, 1, 1.5 and 2% in pilot study)
1 % in this study

No. of animals per group:

not applicable

Control animals:

no

Remarks:

not applicable, because humans

Details on study design:

The application areas were located on the clinically normal skin of the paravertebral mid back. According to the number of different treatment options (see below), 14 test areas with a space of 3 cm between each test chamber were marked with a permanent marker, resulting in 4 vertical rows.

The test areas were randomized among the volunteers in order to avoid an anatomical selection bias. Aliquots of 50 ml of the freshly prepared aqueous irritants were applied to each test area by an occlusive epicutaneous patch test system (Large Finn Chambers® on Scanpor®, 12 mm diameter with filter discs, Epitest Ltd., Hyrlä, Finland). Patches were removed after 30 min. The exposed areas were rinsed with 10 ml of tap water and carefully dried with a paper tissue without rubbing. After a 3-hour interval, a second exposure with one of the irritants, according to the different treatment options, was performed. Using this scheme of application, each test site was repeatedly treated for 4 days.

Details on in vitro test system (if applicable):

not applicable

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Remarks on result:

other: The human volunteer study revealed no irritating effects of DMA on skin; possible processes at barrier are discussed in detail in this publication.

One day of treatment with detergents or biogenic amines did not result in irritation in any of the test sites.

All biogenic amines tested caused barrier disruption. The ranking order was TMA/TMA > DMA/DMA > AM/AM. The sequential irritation of the biogenic amines with SLS (AM/SLS, DMA/SLS and TMA/SLS) resulted in an increase in the barrier disruption starting for all three groups already on day 3, but this barrier disruption was less prominent than SLS/SLS alone.

The irritation was assessed measuring the redness.

The biogenic amines without the combination with SLS (AA/ AA, DMA/DMA, TMA/TMA) did not induce a significant irritation measured by Chromameter a* values. However, the combination with SLS depicted a significant increase in redness values for AM/SLS (only for day 5) and TMA/SLS (from day 3 on), while such an increase in the combination with DMA/SLS was not detectable

The biogenic amines in combination with SLS showed all an increase of the stratum corneum pH, e.g. AM/SLS on day 5, DMA/ SLS and TMA/SLS already on day 4.

The values assessed with the visual score were overall very low.

Conclusions:

The highest irritative potential could be detected, as expected, by the double application of SLS/SLS followed by NaOH/SLS. Next we ranked TMA/SLS, followed by AA/SLS > AM/SLS > DMA/SLS.
Biogenic amines induce a permeability barrier disruption after 3 days of application in a tandem repeated irritation test model. This effect was paralleled with the onset of inflammatory signs and an increase in pH. The sequential application of SLS further increased these effects, and the initiation of both barrier disruption and inflammation occurred earlier.

Executive summary:

In the present study, we were able to show for the first time that biogenic amines cause disruption of the permeability barrier. However, the application of each of the three biogenic amines did not reveal a significant irritation or increase in SC pH. This dichotomy of usually related parameters of barrier disruption and induction of irritation might be based on the fact that the amines disturbed the intercellular barrier-lipid processing but did not induce a pH change and a subsequent increase in pH. Sequential application of SLS further enhanced the barrier disruption induced by the biogenic amines. The only exception was the irritation parameter Chroma a*, where no significant increase of redness could be observed. The TMA/SLS irritation and barrier disruption was slightly more prominent than those induced by AM/SLS and DMA/SLS, which can be explained by the higher concentration (1.5 vs. 1.0%). Since these results are detectable in all analysed parameters, we assume that the described features may be consistent properties of biogenic amines.

We assume that the mechanism by which the biogenic amines induce a barrier disruption and inflammatory reaction are different from that of SLS.

The contact with both classes of irritants however did not show overadditive effects.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The basis for this read-across is the formation of common breakdown product(s) (scenario 1 according to the Read-Across Assessment Framework (RAAF; ECHA 2017). It has been accepted generally that dimethylamine arises from ingested choline via a bacterial degradation product and the intermediate trimethylamine with subsequent demethylation (Asatoor and Simenhof, 1965; Lowis et al., 1985; Zeisel et al., 1983).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
source substance:
choline chloride
structural formula: C5H14ClNO
SMILES: C[N+](C)(C)CCO.[Cl-]
CAS 67-48-1
purity: not specified

target substance:
dimethylamine
structural formula: C2H7N
SMILES: CNC
CAS 124-40-3
purity: ≥ 99.9 %

3. ANALOGUE APPROACH JUSTIFICATION
Only one-third of the ingested choline appears to be absorbed intact. The major part is either oxidized to betaine or metabolized by the intestinal microflora to trimethylamine (De La Huerga, 1953; Zeisel, 1989), whereas the latter is greatest when large amounts of choline are ingested. Trimethylamine is transformed in rather large quantities into dimethylamine (Asatoor and Simenhof, 1965; Lowis et al., 1985; Zeisel et al., 1983). Therefore, choline is a precursor of DMA (Zhang, 1998). The chloride anion released from choline chloride is a naturally occurring element in the human body. Therefore, it is not expected to influence the toxicokinetic behaviour of the substance.
Thus, the target substance dimethylamine is a metabolite of choline. Therefore, it is expected to follow the same toxicokinetic pattern as the source substance choline.

Reason / purpose for cross-reference:

read-across source

Details on excretion:

Administration of Choline 100 mg tor ats: mean excretion of dimethylamine increasing from 37.5 -4- 5.4/µg to 73-5 4- 4.8/µg per mg creatinine (t = 9.3; P < 0.01).
The formation of dimethylamine from ingested choline was confirmed by giving [Me-14C]choline chloride to two rats. The results show the incorporation of a small but significant amount of radioactivity into dimethylamine. In contrast to the findings in the rats given choline orally, there was no significant increase in urinary dimethylamine, when the same dose of choline was administered by intraperitoneal injection. It seemed, therefore, that the conversion of choline to dimethylamine depended upon the activities of the intestinal flora. To test this, two rats were treated with neomycin (100 mg each, twice daily for 4 days) and were then fed the same dose of radioactive choline chloride as used previously. There was no incorporation of radioactivity into dimethylamine, isolated as the DNP-derivative from urine. Since ingested lecithin can be converted to choline by enzymes in the gastrointestinal tract, the excretion of dimethylamine was studied in three rats before and after administration of 1 g egg lecithin. Dimethylamine excretion increased considerably during 48 h, indicating that the phospholipid is another source of dimethylamine.

Metabolites identified:

yes

Details on metabolites:

Dimethylamine is build when choline, lecithin methylamine hydrochloride or trimethylamine hydrochloride were administered to rats.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
In conclusion, urinary dimethylamine is traced to two sources: (a) from dietary choline; (b) from methylamine, by transmethylation, and methionine serves as the methyl donor.
The major part of ingested choline is either oxidized to betaine or metabolized by the intestinal microflora to trimethylamine (De La Huerga, 1953; Zeisel, 1989), whereas the latter is greatest when large amounts of choline are ingested. Trimethylamine is transformed in rather large quantities into dimethylamine (Asatoor and Simenhof, 1965; Lowis et al., 1985; Zeisel et al., 1983). Therefore, choline is a precursor of DMA (Zhang, 1998). The chloride anion released from choline chloride is a naturally occurring element in the human body. Therefore, it is not expected to influence the toxicokinetic behaviour of the substance. Thus, the target substance dimethylamine is a metabolite of choline. Therefore, it is expected to follow the same toxicokinetic pattern as the source substance choline.

Executive summary:

The study performed by Asatoor et al, in 1965, already gives important data concerning the DMA. They investigated whether DMA is genereated in the mamalian body by feeding rats (in cases also administered via injection) with either choline chloride, lecithin, [14C]methylamine, or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) and then analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way. They could prove that DMA is endogenously build out of choline and lecithin. Furthermore TMA is in part metabolised to DMA, which is then excreted mainly via urine. Additionally MMA is partly methylated to forme DMA.

The major part of ingested choline is either oxidized to betaine or metabolized by the intestinal microflora to trimethylamine (De La Huerga, 1953; Zeisel, 1989), whereas the latter is greatest when large amounts of choline are ingested. Trimethylamine is transformed in rather large quantities into dimethylamine (Asatoor and Simenhof, 1965; Lowis et al., 1985; Zeisel et al., 1983). Therefore, choline is a precursor of DMA (Zhang, 1998). The chloride anion released from choline chloride is a naturally occurring element in the human body. Therefore, it is not expected to influence the toxicokinetic behaviour of the substance. Thus, the target substance dimethylamine is a metabolite of choline. Therefore, it is expected to follow the same toxicokinetic pattern as the source substance choline.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data. Please refer also to the detailed read-across justification attached in section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Dimethylamine and dimethylammonium chloride belong to the group of secondary aliphatic amines with two methyl groups attached to the nitrogen atom (common structure / functional group).
The basicity of amines increases with the length of the aliphatic rest due to electron releasing properties of alkyl groups: the higher the pKa value, the weaker the acid, so the stronger the base.
The solvation of both Dimethylamine and dimethylammonium chloride in water results in solutions of the dimethylammonium cation (“common breakdown product", scenario 1 of the Read Across Assessment Framework (ECHA 2017)). Thus, one must only regard the properties of the respective counterion (“non-common compound”).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance:
dimethylamine hydrochloride
structural formula: C2H7N.ClH
SMILES: [Cl-].C[NH2+]C
CAS 506-59-2
purity: not specified

target substance:
dimethylamine
structural formula: C2H7N
SMILES: CNC
CAS 124-40-3
purity: ≥ 99.9 %

3. ANALOGUE APPROACH JUSTIFICATION
Dimethylamine and dimethylammonium chloride belong to the group of secondary aliphatic amines. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987).
The dissociation constants of DMA allow the conclusion that virtually all molecules of dimethylamine - when dissolved in an excess of water are present as the dimethylammonium cation. Moreover, the available data of DMA with hydrochloric acid shows clearly that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids. Only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both dimethylamine and dimethylammonium chloride in water would result in solutions of the dimethylammonium cation (“common breakdown product"). Therefore, one must only regard the physico-chemical properties of the respective counterion. Dimethylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the dimethylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant (eco)toxicological effects. Both anions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions.
The corrosive effects of DMA can certainly be explained by the high concentration of hydroxyl anions in DMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. DMA (gas) is legally classified as Skin Irrit. 2 and Eye Dam. 1, DMA (aqueous solution) is legally classified as Skin Corr. 1B; DMA-HCl has no legal classification. There is no data available on skin and eye irritation of DMA-HCl. However, DMA-HCl is expected to be minor irritating when compared to DMA, because of the pH neutralisation caused by HCl. Consequently, the enhanced absorption of DMA compared to DMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
Besides the influence of HCl on the pH value of an aqueous solution, it does not bear a relevant intrinsic property, allowing one in general to focus on the dimethylammonium cation.
The similar findings (refer to data matrix outlined below) for both substances support the conclusion that the identical molecule will be formed from both substances when applied systemically, and this molecule, i.e. the dimethylammonium cation, is responsible for the observed effects. In consequence, the dimethylammonium cation is what is left to be considered in both cases and similar effects can be reasonably expected when testing DMA-HCl for the lacking endpoints, compared to the data obtained with DMA. Hence, DMA-HCl may perfectly serve as read-across substance for DMA and vice versa. So, the available data on DMA-HCl can be used to cover all systemic endpoints currently lacking from DMA, making further testing obsolete.

4. DATA MATRIX
There is mainly data available on the toxicological properties of DMA. Data on DMA-HCl covers merely the toxicokinetic endpoint. Hence, the identification and discussion of common properties of DMA and DMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (DMA is as a pure substance, gaseous at room temperature, DMA-HCl is a solid secondary ammonium salt) triggers some differences in the physico-chemical properties like melting point, boiling point, decomposition temperature and vapour pressure. Nevertheless, regarding the application of both substances, i.e. their distributed form, the gaseous character of DMA becomes less relevant as the substances are usually not applied in their pure forms but rather as aqueous solutions.
The available data for the following physico-chemical properties, which are relevant for absorption into living organisms, are very similar. Both substances are small molecules with a molecular weight of 45.0837 (DMA) resp. 80.5367 (DMA-HCl), they are both very soluble in water (50 g/L at 20°C (DMA) and 3000 g/L at 20°C (DMA-HCl)), have a negative logPow (-0.274 (aqueous solution, 25°C, pH 10.8 - 11.1) (DMA) and -3.28 (DMA-HCl)), and at least DMA is readily biodegradable, making it very like for DMA-HCl to bear the same property. Although being expected to be hydrolytically stable in the natural environment, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, DMA has a pKa of 10.73 at 20°C, which indicates that dimethylamine exists almost entirely in the cationic form at pH values of 5 to 9.

Reason / purpose for cross-reference:

read-across source

Preliminary studies:

no data

Type:

excretion

Results:

GFR = 2.5 mg/min

Type:

excretion

Results:

renal plasma flow rate (RPF) = 10 mL/min

Type:

excretion

Results:

renal blood flow rate (RBF) = 20 mL/min

Type:

excretion

Results:

apparent total clearance = 9-10 mL/min

Details on absorption:

no data because administered via injection

Details on distribution in tissues:

The mean blood peak concentration of labeled DMA achieved following the 2-µmol dose was comparable to the concentration of endogenous DMA, which was found to be 2.5 ± 0.2 nmol/mL (n = 5). Except for the earliest (15 min) time point, the data for any given dose fell on a straight line, as predicted by the one-compartment, linear model. The lines shown are the least-squares fits to the data for / > 30 min. However, contrary to expectations for one-compartment, linear behavior, the intercepts and slopes of these lines depended on D. Thus, the apparent volume of distribution and clearance were dose dependent. As the dose of DMA injected was increased, there were decreases in the calculated values of both VD and k. The magnitude of these decreases was such that, in general, there was no overlap of the 90% confidence intervals for these parameters for the different doses.

The tissue-to-blood concentration ratios measured 1 hr following low doses of [14C]DMA or [14C]TMA tended to exceed those in blood, sometimes by large factors. The tissue-to-blood ratios for DMA were typically ~2, with a value of 8 in kidney.

The volumes of distribution for DMA and TMA in similar-size rats are 2260 and 725 mL, respectively.
From the percentage weight gain over the 5-day period, changes in VD of 40 mL/24 hr for DMA and 7 mL/24 hr for TMA or TMAO were estimated. This implies increases in total body content of DMA, TMA, and TMAO of 0.3, 1.1, and 4.5 µmol/ kg body wt/24 hr, respectively.

Details on excretion:

For DMA, < 1% of the dose was recovered in each of feces, blood, lung, and kidney, while —2% of the radioactivity remained in the liver. The largest portion of the dose, 96%, was recovered in the urine in the first 24 hr following the dose. (Urinary recovery between 24 and 72 hr was found to be negligible.) The total recovery was complete (101%) (Table1)
For both DMA and TMA, — 1% of the dose was recovered in exhaled air as 14C02, while a negligible amount was exhaled as the amine. These results demonstrate that both fecal and respiratory excretion of methylamines in normal rats are negligible. Retention of either the original methylamines or any carbon-based metabolites in tissues is also negligible.

Recovery of the DMA or TMA radiolabel was essentially complete at both dose levels. The urinary recoveries shown for the stable isotopes include not only the administered amine, but also the other two amines assayed by GC/MS. Despite inclusion of all three methylamines, the stable isotope recoveries were substantially lower than those of the radioisotopes. Comparing the recoveries of 100-µmol doses measured with radioisotopes or stable isotopes, the lower values with the latter suggest that significant amounts of the urinary radioactivity were in the form of nonmethylamine metabolites (-20% for DMA and -40% for TMA).

When the DMA stable isotope was administered, there were no detectable amounts of TMA or TMAO stable isotopes in the urine.

Test no.:

#1

Toxicokinetic parameters:

other: VD 2258 mL (at 2 µmol), 1041 mL (at 100 µmol), 375 mL (at 1000 µmol)

Test no.:

#2

Toxicokinetic parameters:

other: k = 10.1 mL/min ( at 2 µmol), 9.3 mL/min (at 100 µmol), 4.1 mL/min (at 1000 µmol)

Metabolites identified:

not measured

Details on metabolites:

The concentration of endogenous DMA = 2.5 ± 0.2 nmol/mL.
DMA is excreted mainly via urine. The applied method did not allow identifying metabolites.
Thesis: at the intermediate dose level ~20% of the DMA appeared in urine as metabolites

Endogenous and Bacterial Synthesis: much less DMA was excreted by the germ-free rats than by normal rats. Since dietary intakes of DMA were comparable in the two groups, this implies that there was a great deal of DMA synthesis by gut bacteria in the normal rats. The calculations show that there may have been a small amount of net endogenous synthesis of DMA, but net bacterial synthesis was about four times greater.

TABLE 1 Radioisotope Balances for DMA and TMA in Normal Rats^a
	DMA	TMA
Feces	0.6 ± 0.1 (5)	0.8 ± 0.03 (5)
Blood	0.5 ± 0.2 (5)	0.04 ± 0.005 (5)
Lung	0.3 ± 0.04 (5)	< 0.001 (5)
Liver	2.3 ± 1.2 (5)	0.02 ± 0.004 (5)
Kidney	0.5 ± 0.05 (5)	0.003 ± 0.001 (5)
Urine	96 ± 2 (2)	96 ± 3 (2)
MA trap^b	0.03 ± 0.01 (2)	0.05 ± 0.01 (2)
CO2	1 ± 0.2 (2)	0.8 ± 0.06 (2)
Total^c	101 ± 2	98 ± 3
^aValues are percentages of dose recovered, expressed as means ± SE with the number of measurements (number of rats) in parentheses. ^bMA, methylamine. ^cError estimates for the totals were computed from the standard errors (SE) of the other entries using

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
DMA is mainly excreted via the urinary tract and bears the some potential to circulate between tissues and the gastro intestinal tract. The target substance dimethylamine and the source substance dimethylammonium chloride used in this study belong to the group of secondary aliphatic amines. The solvation of both, dimethylamine and dimethylammonium chloride in water results in solutions of the dimethylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the dimethylammonium cation. The dimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms.
Therefore both substances are expected to follow the same toxicokinetic pattern.

Executive summary:

The study performed by Smith et al. in 1994, dealt with the metabolic fate of DMA or TMA after injection to rats. There are three sources of dimethylamine (DMA): diet, bacterial synthesis, and endogenous synthesis. Fish contains significant quantities of DMA and TMA, and is probably a major dietary source of methylamines. As TMAO is an end-product of nitrogen metabolism in fish, it can be metabolized to DMA and TMA by bacteria once fish have been killed. The endogenous concentration of DMA, was found to be 2.5 +/- 0.2 nmol/mL. So DMA enters the body from intestinal absorption of both dietary DMA and DMA formed by bacterial action in the lower gut or after endogenous synthesis after conversion of choline to DMA (see also Asatoor et al. 1965) and leaves by urinary excretion. The much greater urinary excretion of DMA by normal rats than by germ-free rats is clear evidence for significant synthesis of DMA by gut bacteria. Urine analysis following doses of stable isotopes showed also that DMA was not converted to TMA or TMAO. There is also some endogenous synthesis of DMA, possibly from monomethylamine (MMA). So the results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes.

When dimethylamine hydrochloride (DMA-HCl) is administered in vivo intravenously or intraperitoneally it is readily absorbed (Smith, 1994). After the uptake it is uniformly distributed in the body, interestingly with the volume of distribution exceeding the animal size, suggesting that it is highly concentrated at one or more locations in the body (Smith, 1994). The tissue-to-blood concentration ratio of 8 found for DMA in kidney greatly exceeds values for other body compartments, including gastric fluid (Smith et al., 1994). The apparent volume of distribution and clearance were dose-dependent. About 20 % of the administered DMA appear in the urine as non-methylamine metabolites.The experiments revealed, that the only important pathway for elimination of the three methylamines (and their metabolites) tested is urinary excretion. Fecal excretion, exhalation in breath, and retention in tissues are all negligible in normal rats. The experiments conducted showed that DMA is avidly secreted by the renal tubules, or there might exist as an alternative explanation for the dose dependence of the volume of distribution an active transport of DMA or TMA from extracellular to intracellular fluid. The results strongly suggest that there are various regions outside the gastrointestinal contents which contain high concentrations relative to the blood of these methylamines.

The target substance dimethylamine and the source substance dimethylammonium chloride used in this study belong to the group of secondary aliphatic amines. The solvation of both, dimethylamine and dimethylammonium chloride in water results in solutions of the dimethylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the dimethylammonium cation. The dimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms.

Therefore both substances are expected to follow the same toxicokinetic pattern.

Description of key information

Short description of key information on bioaccumulation potential result:
Smith, Wishnok, Deen, 1994, toxicology and applied pharmacology 125, 296-308, administration of DMA intraperitoneally to rats and investigation of the distribution, metabolism and excretion.
Mitchell et al. 1994, Xenobiotica, 1994, Vol. 24 No. 12, 1215-1221, administration of DMA orally to rats and mice and investigation of the distribution, metabolism and excretion via radiolabelling.
Ishiwata et al., 1984, IARC scientific publications / World Health Organisation, International Agency for Research on Cancer, administration of DMA orally via diet to rats and investigation of the distribution, metabolism and excretion.
Zeisel and DaCosta, 1986, Cancer Research 46, 6136-6138 December 1986, investigation of methylamine excretion after ingestion of fish containing methylamines in humans.
Asatoor et al., 1965, Biochimica et biophysica acta, 111 (1965) 384-392, administration of choline chloride (intraperitoneal) or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) [14C]methylamine (intraperitoneal) or L[Me-14C]Methionine (intraperitoneal) or 14C-MMA to rats (orally or intraperitoneally) and analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way.

Short description of key information on absorption rate:
Fluhr, 2005, Additive Impairment of the Barrier Function and Irritation by Biogenic Amines and Sodium Lauryl Sulphate: A Controlled in vivo Tandem Irritation Study, Skin Pharmacol Physiol 2005;18:88–97, 1 % DMA in distilled water on human skin (paravertebral mid back).
No significant irritation, but barrier disruption of the stratum corneum was observed.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

The metabolism, disposition and toxicokinetics of dimethylamine have been well characterized. First of all, it is important to take into account, that dimethylamine has three different sources: the diet (for example in fish), bacterial synthesis in the intestinal tract and endogenous synthesis. As TMAO is an end-product of nitrogen metabolism in fish, it can be metabolized to DMA and TMA by bacteria once fish have been killed. The endogenous concentration of DMA was found to be 2.5 ± 0.2 nmol/mL.The much greater urinary excretion of DMA by normal rats than by germ-free rats is clear evidence for significant synthesis of DMA by gut bacteria. In particular, DMA can be build in the body during bacterial degradation of choline or lecithin inside the intestinal tract (DMA excretion was directly related to the dose of choline administered), but variations in choline intake that might occur as part of consumption of normal foods are not likely to significantly increase TMA and DMA production, and therefore change our exposure to precursors of nitrosamines only somewhat. Additionally monomethylamine can be methylated to DMA. In total the knowledge of the endogenous occurrence influences mainly the categorization and classification of dimethylamine.

In rats and in mice, dimethylamine and dimethylamine hydrochloride were rapidly absorbed, uniformly distributed (interestingly the volume of distribution exceeds the animal size, so it has to be stated, that DMA must accumulate in some tissues more than in others) and eliminated mainly unchanged (about 80 %) via urine in both sexes of rats (only negligible amount are excreted via feces or exhaled). The experiments conducted showed that DMA is avidly secreted by the renal tubules. About 20 % of the administered DMA appear in the urine as methylamine (3 -5 %) and non-methylamine metabolites. Nevertheless there exists also the possibility of DMA being metabolized to N-Nitrosodimethylamine, which is a potent carcinogen. It is not possible to exclude the possibility of nitrosamine formation, but it seems rather unlikely that a significant amount of nitrosamine can be built, since this reaction occurs mainly at low pH, like in the stomach and requires nitrite. For example; it was estimated that, if a man ate a 300-g meal containing 12 mg dimethylamine hydrochloride and 60 mg sodium nitrite, not more than about 3 µg DMN might be expected to be formed intragastrically. This dose ingested daily may be too low to be significantly carcinogenic, though it should be stressed that even tiny incidences may assume importance in a large population.

Interestingly, DMA secreted from the blood into the small intestine can be re-absorbed. So the intestinal DMA concentration is a balance between absorption, secretion and formation and the amount of intestinal moisture.

Based on the results obtained in these studies, dimethylamine may be considered to be a mostly in the intestine, rapidly absorbed, and well eliminated (mainly unchanged) substance in rats, mice and humans. It bears the possibility to be metabolized to the carcinogenic substance N-Nitrosodimethylamine.

Discussion on bioaccumulation potential result:

The study performed by Smith et al. in 1994, dealt with the metabolic fate of DMA or TMA after injection to rats. There are three sources of dimethylamine (DMA): diet, bacterial synthesis, and endogenous synthesis. Fish contains significant quantities of DMA and TMA, and is probably a major dietary source of methylamines. As TMAO is an end-product of nitrogen metabolism in fish, it can be metabolized to DMA and TMA by bacteria once fish have been killed. The endogenous concentration of DMA, was found to be 2.5 ± 0.2 nmol/mL. So DMA enters the body from intestinal absorption of both dietary DMA and DMA formed by bacterial action in the lower gut or after endogenous synthesis after conversion of choline to DMA (see also Asatoor et al. 1965) and leaves by urinary excretion. The much greater urinary excretion of DMA by normal rats than by germ-free rats is clear evidence for significant synthesis of DMA by gut bacteria. Urine analysis following doses of stable isotopes showed also that DMA was not converted to TMA or TMAO. There is also some endogenous synthesis of DMA, possibly from monomethylamine (MMA). So the results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes. When dimethylamine hydrochloride (DMA-HCl) is administered in vivo intravenously or intraperitoneally it is readily absorbed (Smith, 1994). After the uptake it is uniformly distributed in the body, interestingly with the volume of distribution exceeding the animal size, suggesting that it is highly concentrated at one or more locations in the body (Smith, 1994). The tissue-to-blood concentration ratio of 8 found for DMA in kidney greatly exceeds values for other body compartments, including gastric fluid (Smith et al., 1994). The apparent volume of distribution and clearance were dose-dependent. About 20 % of the administered DMA appear in the urine as non-methylamine metabolites.The experiments revealed, that the only important pathway for elimination of the three methylamines (and their metabolites) tested is urinary excretion. Fecal excretion, exhalation in breath, and retention in tissues are all negligible in normal rats. The experiments conducted showed that DMA is avidly secreted by the renal tubules, or there might exist as an alternative explanation for the dose dependence of the volume of distribution an active transport of DMA or TMA from extracellular to intracellular fluid. The results strongly suggest that there are various regions outside the gastrointestinal contents which contain high concentrations relative to the blood of these methylamines.

Another Study by Mitchell et al, 1994, confirmed the results achieved by Smith et al, 1994. In both rodents DMA was rapidly absorbed in the small intestine, caecum and large intestine and is distributed rapidly through the blood and then disappeared rapidly into the urine, which was the major route of excretion with the majority of radioactivity (91 %) being voided during the first day (Mitchell et al, 1994). Only a small proportion was secreted in the intestine and bile. Interestingly DMA secreted from the blood into the small intestine can be re-absorbed. So the intestinal DMA concentration is a balance between absorption, secretion and formation and the amount of intestinal moisture.

The majority of [14C]-dimethylamine administered was excreted unchanged in the urine with only small amounts being demethylated to [14C]-methylamine. In all 0 -24 h urine samples examined two radioactive areas were found, which corresponded to authentic methylamine and dimethylamine (little variation between the species with 96 -6 ± 2 -5 and 95 5 ± 1 -3 % of the radioactivity being present as dimethylamine in rat and mouse respectively. The demethylation product, methylamine, only accounted for 3-4 ± 1-5 (rat) and 4-5 ± 1-3 % (mouse) of the 0-24 h urinary radioactivity output.(Mitchell et al., 1994). Additional small amounts of radioactivity were observed in the 24-72 h urine (2 %), in feces (2 %) and in exhaled air (1 %), with a further quantity (1 %) being detected within the carcass after 3 days. Good overall recoveries were achieved; suggesting that only trace amounts of volatile compounds had been exhaled and escaped detection.

Ischiwata et al. examined 1994 the absorption, distribution and excretion of DMA in wistar rats. The fate of DMA in rats is very little or no absorption in the stomach but rapid absorption in the intestine - especially in the upper part of the small intestine - secretion of some of the compounds from the blood into the saliva or the digestive tract, and excretion of most of an ingested or otherwise administered dose of either compound in the urine (Ishiwata et al., 1984).

Zeisel and DaCosta reported that DMA excretion in humans was more than 4 times greater on the day that methylamine-containing fish was eaten than on the control day. (Zeisel and DaCosta, 1986). Human subjects (n = 5) ingested a diet of known methylamine content for 2 days. On Day 3, they ate fish at the luncheon and dinner meals. On Day 4, they again ate the control diet. A single portion of fish contained as many methylamines as were normally excreted by the human in 2 days. Dimethylamine excretion increased more than 4-fold (from 5.6 to 24.1 µmol/24h/kg of body weight).

In humans, DMA is absorbed after eating fish with an increased level of methylamines (DMA and TMA). With respect to the metabolism of DMA the following conclusion can be drawn: DMA is readily absorbed via oral route (Zeisel, 1986). After ingestion of fish with elevated levels of methylamines the DMA excretion via urine is elevated (2.8-fold). Most of the increase in urine methylamine excretion was in the form of DMA.

The study performed by Asatoor et al, in 1965, already gives important data concerning the DMA. They investigated whether DMA is generated in the mammalian body by feeding rats (in cases also administered via injection) with either choline chloride, lecithin, [14C]methylamine, or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) and then analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way. They could prove that DMA is endogenously built out of choline and lecithin. Furthermore TMA is in part metabolized to DMA, which is then excreted mainly via urine. Additionally MMA is partly methylated to form DMA. So these results are in accordance with the results obtained by Ischiwata et al. in 1984.

Zeisel an Coworkers investigated in 1989 the fate of via orogastric intubation administered choline in the rat intestine (Spraque-Dawley). With the help of radiolabeled choline its uptake and its metabolism were investigated in the intestines. Additionally the metabolites excreted via urine were determined. They found that choline increased the excretion of TMA and TMAO and increased DMA excretion. The absorption mechanisms for choline has a high capacity (most of the choline was absorbed). At the low dose, choline-derived radiolabel was absorbed from the intestinal lumen before it reached the areas of gut colonized with bacteria. At the high dose of choline, much more label reached the colon, and they suggest that an appreciable portion of the choline-derived radiolabel that was absorbed did so via diffusion across the colon. In terms of absolute amounts, much more choline-derived radiolabel was present in the colon after the high dose. They believe that the disproportionate rise in TMA and TMAO excretion observed at the higher choline dose occurred because choline reached the bacterially colonized large intestine in appreciable quantities. The capacity for TMA oxidation must be very large, since TMAO excretion could increase over a thirtyfold range. Despite this capacity, significant amounts of TMA (approximately 10 % of the TMA that must have been formed prior to conversion to TMAO) escaped this fate and was excreted intact. It has been suggested that the major precursor of DMA is the TMA formed by gut bacteria. The rate of increase in DMA excretion with increasing choline dose was different from the rate of increase in TMA excretion at the lower doses of choline. The amount of TMAO that was formed suggests that increasing amounts of TMA could have been transiently available for DMA synthesis as choline doses were increased. At the higher choline dose there was no jump in DMA excretion, but there was a large increase in TMA and TMAO excretion. This suggests that formation of DMA was not tightly linked to the availability of TMA. The data show that DMA excretion was directly related to the dose of choline administered, suggesting that choline, or a metabolite of choline, is likely to be a precursor of DMA. Previously, they found that the presence of gut bacteria was not essential for the formation and excretion of DMA in rats, while gut bacteria made a significant contribution to TMA excretion.

In summary, they have found that there is a critical concentration of choline which must be achieved within gut lumen before choline absorption processes in the small intestine are overloaded, thus allowing sufficient choline to reach the large intestine. Since it is the large intestine which is colonized with bacteria, large doses of choline are, therefore, much more likely to be converted by bacteria to TMA. The data suggest that variations in choline intake that might occur as part of consumption of normal foods are not likely to significantly increase TMA and DMA production, and therefore are not likely to greatly change our exposure to precursors of nitrosamines. Dietary intake of preformed DMA and TMA and endogenous production of these amines are likely to be far more significant sources.

In 1998, Zhang et al. investigated the excretion of DMA after administration of various related amine precursors. They found trimethylamine N-oxide (at a dose rate of 1 mmol/kg body weight) to be 20 times more efficient at providing dimethylamine than an equivalent dose of choline. Additionally less than 1 % of the choline dose was converted to dimethylamine. Slightly more of an equivalent trimethylamine dose (1.6 %) was converted to dimethylamine. So trimethylamine N-oxide is undoubtedly a significant dietary source of dimethylamine and its carries the potential of being converted to carcinogenic nitrosodimethylamine. However, other dietary sources of dimethylamine probably lie undetected amidst the myriad of anutrient chemicals within foodstuffs, and the continued excretion of small amounts of dimethylamine from antibiotic treated and germfree rats (Asatoor et al., 1967; Zeisel et al., 1985) implies that dietary components, although probably the major source, are not the only originator of dimethylamine.

In 1993, Gut et al, investigated the in vitro oxidation of trimethylamine (TMA) to TMA N-oxide (TMAO) and dimethylamine (DMA) in rat liver microsomes. In details the role of flavin-containing monooxygenase and cytochrome P450 in the N-oxidation and N-demethylation of TMA was studied. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16 alpha-carbonitrile had little or no effect on the liver microsomal metabolism of TMA to TMAO or DMA. Changing the atmosphere in the incubation vessel from 20 % oxygen/80 % nitrogen (air) to 100 % oxygen had a selective stimulatory effect on the N-oxygenation of TMA but did not affect TMA N-demethylation. In addition, the Km for TMA N-demethylation was 5-fold higher than for the N-oxygenation reaction. The results of these studies suggest that the enzyme systems responsible for N-demethylation and N-oxygenation are different and that they are under different regulatory control. Carbon monoxide (CO/O2 = 80/20) had little or no inhibitory effect on either the N-demethylation or N-oxygenation of TMA by liver microsomes from control or pregnenolone 16 alpha-carbonitrile- treated rats. Additional studies indicated that methimazole, an inhibitor of FAD-containing monooxygenase (FMO), was a potent inhibitor of TMA oxidation. Evidence was presented that FMOs are the major enzymes responsible for N-demethylation and N-oxygenation of TMA in rat liver microsomes and that cytochrome P450 enzymes do not play a major role in the metabolism of TMA to TMAO or DMA. But since N-demethylation of tertiary amines is an unusual reaction for FMO, a possibility of another thermolabile enzyme catalyzing N-demethylation of TMA cannot be excluded entirely.

Mirvish et al. reinvestigated in 1970 the dimethylamine nitrosation, with the aid of tritiated dimethylamine labeled in the methyl groups, and they give estimates of DMN formation in the stomach and during food storage. Since nitrosation occurs most readily under acidic conditions, DMN and other nitrosamines could also be produced in the gastric contents during digestion. At pH 3.4, the rate of dimethylnitrosamine (DMN) formation was proportional to the dimethylamine concentration and to the square of nitrite concentration and the rate of reaction was maximal. The rate constants were used to estimate the amount of DMN expected to be formed in the gastric contents after ingestion of food containing various concentrations of dimethylamine and nitrite and during storage of such food. If the amine and nitrite concentrations are raised while remaining equal to each other, DMN formation should increase with the cube of reactant concentration.

For example; it was estimated that, if a man ate a 300-g meal containing 12 mg dimethylamine hydrochloride and 60 mg sodium nitrite, not more than about 3 µg DMN might be expected to be formed intragastrically. This dose ingested daily may be too low to be significantly carcinogenic, though it should be stressed that even tiny incidences may assume importance in a large population.

In conclusion, it should be possible theoretically to induce liver tumors in rats by feeding high doses of dimethylamine and nitrite, despite the negative results obtained by earlier workers. Nitrosamine formation should be suspected particularly in foods stored under mildly acidic conditions. The formation of nitrosamines other than DMN could be a more serious problem than that of DMN itself, since the rate of nitrosation increases 1000-fold as the basicity of the amine decreases on proceeding from dimethylamine to aromatic amines such as N-methylaniline.

Discussion on absorption rate:

In the study performed by Fluhr et al, 2005, it was shown that biogenic amines cause disruption of the permeability barrier. However, the application of each of the three biogenic amines did not reveal a significant irritation or increase in SC pH. Sequential application of SLS further enhanced the barrier disruption induced by the biogenic amines. The only exception was the irritation parameter Chroma a*, where no significant increase of redness could be observed. The TMA/SLS irritation and barrier disruption was slightly more prominent than those induced by AM/SLS and DMA/SLS, which can be explained by the higher concentration (1.5 vs. 1.0 %). Since these results are detectable in all analyzed parameters, we assume that the described features may be consistent properties of biogenic amines. They assume that the mechanism by which the biogenic amines induce a barrier disruption and inflammatory reaction are different from that of SLS. The contact with both classes of irritants however did not show over additive effects.