4,4'-methylenebis[2,6-diethylaniline]

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics, other

Type of information:

other: expert assessment based on available toxigolocial data

Adequacy of study:

weight of evidence

Study period:

January 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: expert assessment based on available toxigolocial data

Reason / purpose for cross-reference:

assessment report

Objective of study:

toxicokinetics

Principles of method if other than guideline:

expert assessment based on available toxigolocial data

GLP compliance:

no

Radiolabelling:

no

Metabolites identified:

not specified

M-DEA: assessment of TK profile

There are no specific TK data for4,4′-methylenebis(2,6-diethylaniline) (M-DEA). Two approaches have been evaluated for filling this data gap: (1) read-across to a structurally-related substance, diethyltoluenediamine (DETDA), for which TK data are available; and (2) an assessment based upon toxicology data for M-DEA itself.

Read-across to DETDA

Read-across from a source substance to a target substance requires sufficient structural and physicochemical similarities between the two that the data for the source substance can be reliably considered to be relevant to the target substance. The structures and relevant available physicochemical data for M-DEA and DETDA are given below.

	M-DEA	DETDA (as DETDA 80)
	4,4′-methylenebis(2,6-diethylaniline)	3,5-diethyltoluene-2,4-diamine 3,5-diethyltoluene-2,6-diamine
Molecular weight	310.48	178.27
Melting point	87–89°C	-6°C
Water solubility (20°C)	0.00044 g/L	0.0227 g/L
Log Pow(20°C)	4.4	1.38

Structural considerations

The core structure of M-DEA is of two aromatic rings joined by a methylene bridge. Each ring has an amine moiety in theparaposition, which has two proximal (ortho) ethyl groups.

DETDA consists of two isomers, each of a single aromatic ring with two amine moietiesmetato each other. In one isomer (3,5-diethyltoluene-2,4-diamine), one amine group has two proximal ethyl groups, while the other has proximal ethyl and methyl groups. The amine groups of the second isomer (3,5-diethyltoluene-2,6-diamine) both have proximal ethyl and methyl groups.

Therefore, both M-DEA and DETDA contain two amine groups, but in M-DEA both of these are bounded by two ethyl groups while in DETDA at least one amine has a proximal methyl group. Furthermore, M-DEA is a larger molecule, with two dialkylated aromatic rings that are expected to increase lipophilicity.

Physicochemical considerations

As expected from the higher ratio of hydrophilic groups to overall molecular weight, DETDA has substantially higher water solubility than M-DEA. More importantly, its Log P_owis much lower (1.38 vs. 4.4).

Overall evaluation

M-DEA is substantially more lipophilic than DETDA. This will affect both absorption by all routes of exposure, as well as distribution within the body. Alkyl substituents that lieorthoto an aromatic amine moiety reduce the metabolism of that moiety, which becomes more substantial with increasing chain length, i.e. ethyl>methyl (Glendeet al., 2001). Therefore, the amine substituents in M-DEA may be less susceptible to metabolism than at least the methyl/ethyl bound amine substituent(s) in DETDA, although the extent of any difference is not possible to judge. The differences in fat deposition and primary/secondary metabolism of M-DEA compared to DETDA will affect its relative excretion via the urine and faeces.

Overall, therefore, it is concluded that DETDA is not an appropriate read-across source for M-DEA.

Toxicology of M-DEA

M-DEA is not corrosive or irritating to the skin of rabbits following 4 hours of occlusive exposure. The LD50 following oral administration to the rat is 1901 mg/kg; a dose of 2000 mg/kg resulted in 5/5 deaths in males and 2/5 deaths in females. The principal clinical signs were lethargy; prostration; hunched posture; and staining around the eyes, nose, and mouth. Surviving animals were free of symptoms 7 to 11 days after dosing. In an acute toxicity test by dermal administration, 0/5 male and 0/5 female rats died, or showed any adverse clinical signs of treatment, including irritation, at the single dose of 2000 mg/kg.

Repeated dose toxicity data are available from a 90-day study, and a 14-day dose range-finding study. In the 14-day study, M-DEA was administered by gavage at dose levels of 25, 50, or 100 mg/kg bw/day to groups of male and female rats. M-DEA induced significant liver weight increase in males and females at all dose levels, associated with increased activity of γ-glutamyltransferase (GGT) at the highest dose, and elevated serum cholesterol in females at all dose levels, associated with cytoplasmic vacuolation in the hepatocytes at the mid- and high-dose levels. In the 90-day study, M-DEA was similarly administered at dose levels of 10, 25, or 50 mg/kg bw/day. No clinical signs of treatment were noted during the administration period, although there was a reduction in body weight and body weight gain in animals of the mid- and high-dose groups. Clinical chemistry indicated some effect on liver function; serum GGT, cholesterol, bilirubin, and alanine aminotransferase (ALT) were elevated, while alkaline phosphatase was not. Liver weight increased at all dose levels, with macroscopic and microscopic (cytoplasmic vacuolation) changes to the liver in the mid- and high-dose groups. Similar responses have been observed in pregnant rats that were administered M-DEA by gavage during a prenatal developmental toxicity study.

M-DEA has been evaluated for genetic toxicity in three testsin vitro(chromosomal aberration test in Chinese hamster lung cells, and HPRT gene mutation and micronucleus tests in Chinese hamster ovary cells), in the presence and absence of metabolic activation. Under equivalent conditions, M-DEA was without effect either with or without metabolic activation. Although these reports have been included in this evaluation, they do not allow any conclusions on metabolism of M-DEAin vitro, and thereforein vivo, to be drawn.

Overall assessment

Some conclusions on the TK characteristics of M-DEA may be drawn from studies reported above.

M-DEA was toxic following bolus oral administration. The effects were not the result of local irritation to the gastro-intestinal tract, indicating some degree of systemic exposure by this route of administration. Comparing the results of the oral and dermal acute tests indicates that dermal absorption occurs to a lesser extent, since the median lethal oral dose is lower than the dermal limit no effect dose.

The toxicity of M-DEA appears to increase somewhat with repeated exposure. Since the test material was administered by oral gavage, then changes in food consumption as animals age does not explain this observation. This suggests that either M-DEA (or a relevant metabolite) accumulates in the body, or that repeated exposures result in the induction of enzymes that activate M-DEA to a toxic intermediate. Certainly, the high Log K_owfor M-DEA is consistent with deposition into adipose tissue.

The increase in ALT and bilirubin are indicative of liver damage, and this was confirmed histologically. Interestingly, while the ratio of aspartate transaminase to ALT in the serum was generally unaffected, and even lowered in high-dose males (Figure 1A, 1C), the ratio of GGT to ALT was significantly increased (Figure 1B, 1D). This may indicate an induction of GGT, which in turn suggests possible induction of deactivation pathways related to conjugation with glutathione.

References

Glende C., Schmitt H., Erdinger L., Engelhardt G., Boche G. (2001). Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents. Part I. Alkylationorthoto the amino function.Mutation Research 498:19–37.

Conclusions:

From analysis of toxicity data for M-DEA, the following qualitative TK characteristics are proposed. M-DEA is absorbed from the gastrointestinal tract following oral administration. Deposition will favour lipophilic stores such as adipose tissue. M-DEA, or a further active metabolite, likely undergoes conjugation with GSH, prior to elimination. It is not possible to make quantitative estimations from the available data.

Executive summary:

From analysis of toxicity data for M-DEA, the following qualitative TK characteristics are proposed. M-DEA is absorbed from the gastrointestinal tract following oral administration. Deposition will favour lipophilic stores such as adipose tissue. M-DEA, or a further active metabolite, likely undergoes conjugation with GSH, prior to elimination. It is not possible to make quantitative estimations from the available data.

Description of key information

From analysis of toxicity data for M-DEA, the following qualitative TK characteristics are proposed. M-DEA is absorbed from the gastrointestinal tract following oral administration. Deposition will favor lipophilic stores such as adipose tissue. M-DEA, or a further active metabolite, likely undergoes conjugation with GSH, prior to elimination. It is not possible to make quantitative estimations from the available data.

 

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

There are no specific TK data for4,4′-methylenebis(2,6-diethylaniline) (M-DEA). Two approaches have been evaluated for filling this data gap: (1) read-across to a structurally-related substance, diethyltoluenediamine (DETDA), for which TK data are available; and (2) an assessment based upon toxicology data for M-DEA itself.

Read-across to DETDA

Read-across from a source substance to a target substance requires sufficient structural and physicochemical similarities between the two that the data for the source substance can be reliably considered to be relevant to the target substance. The structures and relevant available physicochemical data for M-DEA and DETDA are given below.

	M-DEA	DETDA (as DETDA 80)
	4,4′-methylenebis(2,6-diethylaniline)	3,5-diethyltoluene-2,4-diamine 3,5-diethyltoluene-2,6-diamine
Molecular weight	310.48	178.27
Melting point	87–89°C	-6°C
Water solubility (20°C)	0.00044 g/L	0.0227 g/L
Log Pow(20°C)	4.4	1.38

Structural considerations

The core structure of M-DEA is of two aromatic rings joined by a methylene bridge. Each ring has an amine moiety in the para position, which has two proximal (ortho) ethyl groups.

DETDA consists of two isomers, each of a single aromatic ring with two amine moieties meta to each other. In one isomer (3,5-diethyltoluene-2,4-diamine), one amine group has two proximal ethyl groups, while the other has proximal ethyl and methyl groups. The amine groups of the second isomer (3,5-diethyltoluene-2,6-diamine) both have proximal ethyl and methyl groups.

Therefore, both M-DEA and DETDA contain two amine groups, but in M-DEA both of these are bounded by two ethyl groups while in DETDA at least one amine has a proximal methyl group. Furthermore, M-DEA is a larger molecule, with two dialkylated aromatic rings that are expected to increase lipophilicity.

Physicochemical considerations

As expected from the higher ratio of hydrophilic groups to overall molecular weight, DETDA has substantially higher water solubility than M-DEA. More importantly, its Log Pow is much lower (1.38 vs. 4.4).

Overall evaluation

M-DEA is substantially more lipophilic than DETDA. This will affect both absorption by all routes of exposure, as well as distribution within the body. Alkyl substituents that lie ortho to an aromatic amine moiety reduce the metabolism of that moiety, which becomes more substantial with increasing chain length, i.e. ethyl>methyl (Glendeet al., 2001). Therefore, the amine substituents in M-DEA may be less susceptible to metabolism than at least the methyl/ethyl bound amine substituent(s) in DETDA, although the extent of any difference is not possible to judge. The differences in fat deposition and primary/secondary metabolism of M-DEA compared to DETDA will affect its relative excretion via the urine and faeces.

Overall, therefore, it is concluded that DETDA is not an appropriate read-across source for M-DEA.

Toxicology of M-DEA

M-DEA is not corrosive or irritating to the skin of rabbits following 4 hours of occlusive exposure. The LD50 following oral administration to the rat is 1901 mg/kg; a dose of 2000 mg/kg resulted in 5/5 deaths in males and 2/5 deaths in females. The principal clinical signs were lethargy; prostration; hunched posture; and staining around the eyes, nose, and mouth. Surviving animals were free of symptoms 7 to 11 days after dosing. In an acute toxicity test by dermal administration, 0/5 male and 0/5 female rats died, or showed any adverse clinical signs of treatment, including irritation, at the single dose of 2000 mg/kg.

Repeated dose toxicity data are available from a 90-day study, and a 14-day dose range-finding study. In the 14-day study, M-DEA was administered by gavage at dose levels of 25, 50, or 100 mg/kg bw/day to groups of male and female rats. M-DEA induced significant liver weight increase in males and females at all dose levels, associated with increased activity of γ-glutamyltransferase (GGT) at the highest dose, and elevated serum cholesterol in females at all dose levels, associated with cytoplasmic vacuolation in the hepatocytes at the mid- and high-dose levels. In the 90-day study, M-DEA was similarly administered at dose levels of 10, 25, or 50 mg/kg bw/day. No clinical signs of treatment were noted during the administration period, although there was a reduction in body weight and body weight gain in animals of the mid- and high-dose groups. Clinical chemistry indicated some effect on liver function; serum GGT, cholesterol, bilirubin, and alanine aminotransferase (ALT) were elevated, while alkaline phosphatase was not. Liver weight increased at all dose levels, with macroscopic and microscopic (cytoplasmic vacuolation) changes to the liver in the mid- and high-dose groups. Similar responses have been observed in pregnant rats that were administered M-DEA by gavage during a prenatal developmental toxicity study.

M-DEA has been evaluated for genetic toxicity in three tests in vitro(chromosomal aberration test in Chinese hamster lung cells, and HPRT gene mutation and micronucleus tests in Chinese hamster ovary cells), in the presence and absence of metabolic activation. Under equivalent conditions, M-DEA was without effect either with or without metabolic activation. Although these reports have been included in this evaluation, they do not allow any conclusions on metabolism of M-DEA in vitro, and therefore in vivo, to be drawn.

Overall assessment

Some conclusions on the TK characteristics of M-DEA may be drawn from studies reported above.

M-DEA was toxic following bolus oral administration. The effects were not the result of local irritation to the gastro-intestinal tract, indicating some degree of systemic exposure by this route of administration. Comparing the results of the oral and dermal acute tests indicates that dermal absorption occurs to a lesser extent, since the median lethal oral dose is lower than the dermal limit no effect dose.

The toxicity of M-DEA appears to increase somewhat with repeated exposure. Since the test material was administered by oral gavage, then changes in food consumption as animals age does not explain this observation. This suggests that either M-DEA (or a relevant metabolite) accumulates in the body, or that repeated exposures result in the induction of enzymes that activate M-DEA to a toxic intermediate. Certainly, the high Log Know for M-DEA is consistent with deposition into adipose tissue.

The increase in ALT and bilirubin are indicative of liver damage, and this was confirmed histologically. Interestingly, while the ratio of aspartate transaminase to ALT in the serum was generally unaffected, and even lowered in high-dose males (Figure 1A, 1C), the ratio of GGT to ALT was significantly increased (Figure 1B, 1D). This may indicate an induction of GGT, which in turn suggests possible induction of deactivation pathways related to conjugation with glutathione.

 

References

Glende C., Schmitt H., Erdinger L., Engelhardt G., Boche G. (2001). Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents. Part I. Alkylationorthoto the amino function.Mutation Research 498:19–37.