Formaldehyde, oligomeric reaction products with aniline

Endpoint summary

Additional information

Justification on read-across of data for the 4,4´-isomer of MDA for oligomeric MDA in the scope of REACH.

Oligomeric MDA (oMDA) is produced by a condensation reaction between aniline and formaldehyde. The main component of the crude product is 4,4’-MDA, which makes up 46 to 65%. Further, the crude product contains minor amounts of methylene-2,4’-dianiline and traces of methylene-2,2’-dianiline. The other main component is a mixture of higher oligomers.

The general molecular formula is:

 H₂N-C₆H₄-[H₂N-C₆H₄]_m-C₆H₄-NH₂

The higher oligomers include mainly 3-ring (m=1; product of 3 aniline + 2 formaldehyde) together with other higher number ring types (m=4-7; 4 to 7 aniline with 3-6 formaldehyde) as shown in the table below.

Following nomenclature for the isoformes are defined:

 

1) CAS name: Benzenamine, 4,4'-methylenebis- (mono constituent substance), CAS number: 101-77-9. EC name: 4,4'-methylenedianiline, EC number:202-974-4

2) CAS name: Formaldehyde, polymer with benzenamine (UVCB), CAS number 25214-70-4. EC name: Formaldehyde, oligomeric reaction products with aniline, EC number: 500-036-1.

Concentration ranges of MDA-isoforms [%]:

CONTENT	4,4’-MDA	Oligomeric MDA
4,4’-MDA	75-100	46-65
2,4’-MDA	0-20	0.8-1.4
2,2’-MDA	0-5	<0.1
Formaldehyde, oligomeric reaction products with aniline	0-15	3- to 7-ring isomers concentrations shown below
3-ring isomers	No data	22-28
4-ring isomers	No data	12-14
5-ring isomers	No data	6-8
6-ring isomers	No data	3-5
7-ring isomers	No data	1-3

2,4'-methylenedianiline: CAS number 1208-52-2, EC number 214-900-8

2,2'-methylenedianiline: CAS number 6582-52-1, EC number 229-512-4

 

Comparison of physico-chemical properties of MDA-isoforms:

Property	4,4’-MDA	oligomeric MDA
Melting range	83 - 92°C	30 - 70°C
MW [g/mol]	198.26	233 (average)
water solubility [g/l]	1.01 (at 25°C)	0.36-1.22 (at 20°C)
logPow(at 25°C)	1.55	1.3 - 2.5
Vapor pressure [hPa]	0.00025 Pa at 25°C	<0.000001hPa at 20°C

 

Since 4,4’-MDA is the main constituent of both isoforms, toxicological properties of the incompletely tested oMDA can be extrapolated from this isomer by means of a worst case consideration. All isomers and homologues have primary aromatic amino groups as a common property.

The leading health effects of 4,4’-MDA are liver toxicity, carcinogenicity and sensitization. These effects may, at least partly, be driven by the bifunctional diamine-structure of the 2-ring molecule in 4,4´-position. In higher-ring compounds it may be predicted that this functionality, at least partly and to an alleviated extent, still exists.

Taking into account the higher degree of saturation of functional groups and the altered physico-chemical properties (higher molecular weight, decreased vapour pressure, decreased water solubility, unchanged / decreased logP_owat physiological pH) a lower reactivity and bioavailability of oMDA can be anticipated.

As a proof of concept data from dated industrial hygiene studies and a chronic study in rats can be used:

·   acute i. p. toxicity in mice demonstrated lower toxicity for oMDA (LD₅₀0,5 g/kg bw, BASF AG 1973) than for 4,4’-MDA (LD₅₀0.147 g/kg bw, BASF AG 1965).

·   acute oral toxicity in rats demonstrated lower toxicity for oMDA (LD₅₀0,7 g/kg bw, BASF AG 1973) than for 4,4’-MDA (LD₅₀0.444 g/kg bw, BASF AG 1975).

·   both isoforms are non irritating on skin and mucosa.

·   subcutaneous injections of 3- and 4- ring fractions of MDA into rats resulted in a significantly lower acute toxicity (LD₅₀2.5 g/kg bw) than 4,4’-MDA (LD₅₀0.2 g/kg bw, Bayer AG 1969). In the same study chronic subcutaneous injections of 3- and 4-ring fractions of MDA were better tolerated than 4,4’-MDA. Life expectancy and tumour incidence of animals injected with a high total dose of 11.2 g/kg of 3- and 4-ring fractions of MDA was approximately identical to the animals injected with a lower total dose of 1.41 g/kg 4,4’-MDA. This trend of decreasing systemic toxicity with increasing number of aromatic rings in the amine molecule was supported by chronic subcutaneous injections of a 8-ring fraction of MDA.

Even though the quality of the cited studies does not comply with current guideline requirements, these considerations support the proposal that 4,4’-MDA can be taken as a reference molecule for oMDA. The data available on 4,4’-MDA can therefore be considered representative for the category for the purpose of hazard evaluation and risk assessment.

 

Absorption:

Following dermal application (2 mg/kg bw onto 2cm² application area) MDA was well absorbed in rats (50% in 96h), though to a minor extend in guinea pigs (29% in 96h) and monkeys (21% in 168h) (El-Hawari et al., 1986). In these experiments, significant amounts of MDA were recovered in the skin of the application area at the end of the observation period (26% in rats and 41% in guinea pigs).

From the percental reduction of total absorbed radioactivity with increased test doses (54% with 0.4 mg/rat to 6% with 4 mg/rat, 17% with 1 mg/guinea pig to 7% with 10 mg/guinea pig), a saturable transport process can be anticipated.

In an occlusive in vitro assay dose-levels of 17.7-40.6 µg/cm²were applied to isolated human skin or 20.1 -34.4 µg/cm² to isolated rat skin, respectively. Absorption rates of 33% with isolated human skin and 13% with isolated rat skin were observed during an observation period of 72h (Hotchkiss et al., 1993). These rates decreased significantly when the application area was not occluded. Like in the in vivo assay major amounts of radioactivity were recovered in the skin surface (25% in rat skin, 17% in human skin).

Distribution:

Following dermal and intravenous application in rats and guinea pigs (El-Hawari et al., 1986) and intraperitoneal application in rats and rabbits (Morgott et al., 1984), MDA was readily distributed. 

The residual radioactivity in the organs following a single i. p. dose of 14C-MDA tends to localize in the liver, kidney, spleen and thyroid at both 24 and 96 hours (Morgott et al., 1984). In addition a significant quantity of radioactivity was detected in plasma and red blood cells. No evidence for accumulation in the body was found, since tissue contents of radioactivity markedly decreased between 24 and 96h after application of the test substance.

Similar distribution patterns were reported following dermal and intravenous application in rats and guinea pigs (El-Hawari et al., 1986), again the liver seems to be the major tissue for recovery of radioactivity.

Excretion:

The fate of MDA seems to vary between different species. Though both, rats and rabbits eliminate at least 90% of an i. p. dose within four days, the primary route of excretion is different for each. Rats excrete approximately 55% of the recovered radioactivity into the feces, whereas fast and slow acetylator rabbits excrete about 80% of the applied compound into the urine and less than 25% into the feces (Morgott et al., 1984). Since the compound was administered by the intraperitoneal route, the amount of fecal radioactivity provided an indication of bilary excretion. These differences in elimination pattern may also be reflected in the resistance of the rabbit to MDA-induced liver damage.

Similar excretion patterns were observed in a comparative study with rats, guinea pigs and monkeys following an intravenous injection (2 mg/kg bw) of 14C-MDA. However, for rats significantly higher urinary excretion 96h following i. v. dosing (67% in urine and 30.7% in feces) was reported compared to i. p. dosing in the study of Margott et al. (1984).

For guinea pigs fecal excretion averaged 56.5% and urinary excretion 35%, whereas in monkeys excretion of 14C-MDA occurred primarily in the urine (84.3%) and to a lesser extent in the feces (9.8%). Peak eliminations occurred within 24h in rats and guinea pigs and within 12h following application in monkeys (El-Hawari et al., 1986).

In the same study elimination following dermal applications of low (2 mg/kg bw) and a high doses (20 mg/kg bw) were described in rats, guinea pigs and monkeys. Fecal/urinary excretion of the applied dose 96h following application averaged 10%/43% for rats and 17%/10% for guinea pigs. In monkeys 1.4% and 19% were detected in feces or urine 168 h following dosing, respectively. The percentage of radioactivity recovered in excreta decreases significantly in the high dose level, again describing a saturable kinetic (El-Hawari et al., 1986).

The total recovery of radioactivity from the rat and slow acetylator rabbit is about 10% less than the recovery from the fast acetylator rabbit. This difference in recovery between fast and slow acetylating rabbits is associated with the greater fecal excretion by the fast acetylator rabbit. 

Metabolism:

Although differences in the quantitative aspects of metabolism remain unelucidated, the in vivo biotransformation pathways of MDA involve N-acetylation reactions as well as an oxidation of the central C-atom and conjugation to glucuronides and sulfates. The N-acetylation apparently represents the major detoxification pathway, whereas the N-hydroxylation additionally being postulated from in vitro studies may lead to potentially toxic intermediates.

Following oral application of MDA to Sprague-Dawley rats (50 mg/kg bw) N-acetyl-MDA has been shown to be the major metabolite (Tanaka et al., 1985). Minor amounts of N, N´-diacetyl-MDA and free MDA were also detected in the urine. The total amount of metabolites added to 3% of the applied dose and was nearly entirely excreted during the 72h observation period.

In a more elaborate analytical assay at least 17 urinary metabolites were characterized upon a single i. p. administration of MDA to Sprague-Dawley rats (30 mg/kg bw) (Morgott, 1984). Mainly, the following acetylated metabolites have been identified: N-acetyl-MDA, N, N´-diacetyl-MDA, N, N´-diacetyl-3-hydroxy-MDA, N-acetyl-4,4' -diaminobenzophenone, and N, N´-diacetyl-4,4´-diamino-benzhydrol.

The study was performed in rats and rabbits, showing that at least 50% of the urinary radioactivity in both species is composed of freely extractable metabolites, which are not glucuronide or sulfate conjugates. Furthermore, O-glucuronide and O-sulfate conjugates only constituted about 3% of total extractable radioactivity, which is approximately 70% in the rat and 80% in the rabbit. However, unlike in the rat, a relatively high percentage of N-glucuronide metabolites were found in rabbit urine.

From the biotransformation products identified in the urine of rats and rabbits it can be seen, that the major biotransformation reactions are N-acetylation and bridge oxidation.

The major urinary metabolite excreted by the rat was N, N´-diacetyl-4,4´-diaminobenzhydrol, which accounted for approximately 40% of the radioactivity in hydrolyzed specimens. The main metabolite in hydrolysed urine of fast and slow acetylator rabbits was the parent compound MDA, which accounted for about 45% and 65% of the radioactivity in hydrolyzed urine specimens, respectively.

The overall metabolism of MDA was extensive compared to the rabbit, where minimal biotransformation occurred.

Binding to macromolecules:

24 hours after a single oral or i. p. administration of MDA a dose dependent increase of hemoglobin-adducts could be detected in the rat (Bailey at al., 1990;). Predominantly, the monoacetylated MDA seems to react with hemoglobin. In contrast to these results Neumann et al. (1993) found more hemoglobin-adducts derived from the parent compound than from N-acetyl-MDA after single oral administration of MDA.