4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics, other

Type of information:

other: Toxicokinetic assessment based on test results of ELINCS-notification

Adequacy of study:

weight of evidence

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Expert assessment based on GLP-studies conducted according to EU-/OECD-guidelines

Objective of study:

toxicokinetics

Qualifier:

no guideline followed

GLP compliance:

no

Radiolabelling:

no

Species:

other: various, see corresponding test results

Strain:

other: various, see corresponding test results

Sex:

male/female

Route of administration:

other: various, see corresponding test results

Vehicle:

other: various, see corresponding test results

Details on exposure:

various, see corresponding test results

Duration and frequency of treatment / exposure:

various, see corresponding test results

Remarks:

Doses / Concentrations:
various, see corresponding test results

No. of animals per sex per dose / concentration:

various, see corresponding test results

Control animals:

no

Positive control reference chemical:

various, see corresponding test results

Details on study design:

various, see corresponding test results

Details on dosing and sampling:

various, see corresponding test results

Preliminary studies:

none

Type:

absorption

Results:

test item will be available for absorption, details see "results" section

Details on absorption:

Although of moderately low molecular weight, P5367 will be physically available for absorption only to a limited extent because at room and body temperature it is a solid with only very limited degree of water solubility. The fat solubility and the high partition coefficient indicate a potential for bioaccumulation.

The results of the oral studies indicate that Lonzacure@ M-CDEA is absorbed from the gastrointestinal tract leading to clinical signs and, at high doses, to mortality. Adaptive liver changes and pathological lesions in the urinary tract observed in the subacute studies indicate that liver metabolism and renal excretion of the parent compound and/or metabolites occur. This is in agreement with the known metabolic and excretion pathways of other aromatic amines in rats.
The results of the acute dermal study did not reveal systemic toxicity. However, due to questionable findings on body weight gains the absorption by this route can not ultimately be excluded.
Inhalation of vapour at room temperature will be negligible based on the low vapour pressure. Although the particle size distribution indicates a limited potential for inhalation, the absorption by the inhalative route is expected to be low based on the low water solubility and the deposition of the particles largely in the nasopharyngeal and tracheobronchial regions from where they may be cleared mechanical retrograde transport mechanisms.

Details on distribution in tissues:

see above

Details on excretion:

see above

Metabolites identified:

not measured

Parameters

Physico-chemical properties

P5367 is an organic intermediate used in technical applications. The material consists of off-white crystals at room temperature, with a melting point of about 88°C and a boiling point in excess of 170°"C which can not however be determined because of decomposition. It has a molecular weight of 380 and is typically > 97.0% pure. P5367 has a water solubility of approximately 25 ug/l at 20°C, a fat solubility of 17000 mg/100 g standard fat HB307 and a partition coefficient of 6.3 (logPow). The vapour pressure is < 0.1 Pa at 25°C, the particle size determination indicate a fraction of about 8.5% with an aerodynamic diameter of less than 10 pm and about 1.1% with less than 2.0 um.

Toxicity

Acute oral toxicity:

The LD50 of P5367 was determined as > 5000 mg/kg using groups of five males and five female rats. One animal only showed clinical symptoms such as emaciation, hunched posture, stained fur and died on day 8. All other animals survived the treatment and were free of symptoms. No gross pathological changes were found in the survivors, the dead animal presented with dark lungs and pale, enlarged liver with dark and yellow patches. No mortality or clinical signs were observed in cats (five males and five females) after a single oral dose of 4000 mglkg.

Acute dermal toxicity:

At a limit dose of 2000 mg/kg no deaths occured in rats There were neither signs of local toxicity nor clinical signs of systemic toxicity Lower than expected body weight gains were observed particularly in females However, in absence of concurrent control values a relation to the treatment remains uncertain.

Subacute (14 day) oral dose range finding study:

Groups of three male and female rats were treated with 0, 1000, 3000 and 10000 ppm P5367 in the diet. Three animals died at the high dose after 6, 12 and 15 days, respectively. Major signs of toxicity were emaciation, bodyweigh loss and lethargy. Liver (males only) and adrenal weights were increased in all groups Pathological changes were seen in high dose animals only and were: hemorrhages of the stomach, bloody content of the urinary bladder and dilation of the urethra and/or renal pelvis.

Subacute (28 day) oral toxicity:

Groups of five male and five female rats were treated with 0, 100, 300 or 1000 ppm P5367 in the diet. Body weight development, feed consumption and feed conversion efficiency were not affected by the treatment. Neither clinical signs nor mortality were observed. Treatment related findings were confined to the high dose and included liver weight increase and hypertrophy of centrilobular hepatocytes. The centrilobular hypertrophy was not accompanied by biochemical evidence of liver damage. No gross lesions were found at terminal necropsy. The no observed effect level (NOEL) was 300 ppm (corresponding to about 38 mglkglday). The high dose level of 1000 ppm (about 138.5 mgkglday) was generally well tolerated.

Metabolism

Mutagenicity -Metabolism:

No indication for mutagenic potential was obtained in a battery of in vitro and in vivo tests. No toxicity was seen in the Ames test both in presence or absence of a rat liver post mitochondria1 fraction (S-9 mix). In the in vitro cytogenetics assay with human lymphocytes P5367 was apparently less toxic in presence of S-9 mix than in its absence.

In vitro metabolism:

The results of an in vitro study with guinea pig liver microsomes indicate that P5367 may be enzymatically hydroxylated to the N-hydroxylamine by a NADPH dependent reaction. The data also suggest that less N-hydroxylamine is metabolically available from P5367 than from the reference substance MOCA.

Assessment

Although of moderately low molecular weight, P5367 will be physically available for absorption only to a limited extent because at room and body temperature it is a solid with only very limited degree of water solubility. The fat solubility and the high partition coefficient indicate a potential for bioaccumulation.

The results of the oral studies indicate that P5367 is absorbed from the gastrointestinal tract leading to clinical signs and, at high doses, to mortality. Adaptive liver changes and pathological lesions in the urinary tract observed in the subacute studies indicate that liver metabolism and renal excretion of the parent compound and/or metabolites occur. This is in agreement with the known metabolic and excretion pathways of other aromatic amines in rats.

The results of the acute dermal study did not reveal systemic toxicity. However, due to questionable findings on body weight gains the absorption by this route can not ultimately be excluded.

Inhalation of vapour at room temperature will be negligible based on the low vapour pressure. Although the particle size distribution indicates a limited potential for inhalation, the absorption by the inhalative route is expected to be low based on the low water solubility and the deposition of the particles largely in the nasopharyngeal and tracheobronchial regions from where they may be cleared mechanical retrograde transport mechanisms.

Conclusions:

See executive summary.

Executive summary:

Although of moderately low molecular weight, P5367 will be physically available for absorption only to a limited extent because at room and body temperature it is a solid with only very limited degree of water solubility. The fat solubility and the high partition coefficient indicate a potential for bioaccumulation.

The results of the oral studies indicate that P5367 is absorbed from the gastro-intestinal tract leading to clinical signs and, at high doses, to mortality. Adaptive liver changes and pathological lesions in the urinary tract observed in the subacute studies indicate that liver metabolism and renal excretion of the parent compound and/or metabolites occur. This is in agreement with the known metabolic and excretion pathways of other aromatic amines in rats.

The results of the acute dermal study did not reveal systemic toxicity. However, due to questionable findings on body weight gains the absorption by this route can not ultimately be excluded.

Inhalation of vapour at room temperature will be negligible based on the low vapour pressure. Although the particle size distribution indicates a limited potential for inhalation, the absorption by the inhalative route is expected to be low based on the low water solubility and the deposition of the particles largely in the nasopharyngeal and tracheobronchial regions from where they may be cleared mechanical retrograde transport mechanisms.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1996 - April 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

other: In vitro investigation of N-hydroxylation of the test substance P5376 in guinea pig liver microsomes and comparison with that of the reference substance MbOCA

Qualifier:

according to guideline

Guideline:

other: In-House

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male

Details on test animals or test system and environmental conditions:

Three young adult male pirbright guinea pigs with a starting body weight of about 400-500 g were housed in the animal facility of the test lab. The guinea pigs were housed in standard cages (macrolon type 4) on softwood bedding at 22°C, at a relative humidity of 25-45%, and a 12 hour light/dark cycle. The animals were acclimatised to laboratory conditions for 8 days prior to sacrifice. A pelleted standard chow and tap water via water bottles were offered ad libitum.
The animals were not fasted prior to necropsy. Upon sacrifice, the animals were bled under carbon dioxide anaesthesia. The livers were immediately chopped, weighed and frozen in liquid nitrogen. Frozen liver samples were stored at -80°C until processed.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Control animals:

no

Details on dosing and sampling:

Subcellular fractionation
Microsomal and cytosolic liver fractions from 3 guinea pigs were prepared individually. The subcellular fractionation was performed at 4°C. The frozen liver samples were thawed to 0°C within 30 min and homogenised in 10 mmol/L TrislHCI buffer, pH 7.5, containing
250 mmol/L sucrose to give a 20% (w/v) homogenate. Microsomal and cytosolic liver fractions were obtained by centrifugation of 12'000g (15 min) supernatants for 1 hour at 100'000g. Microsomes were washed once by resuspending the 100'000g pellets in 50 mmol/L. TrislHCI buffer, pH 7.5, and centrifugation at 100'000g for 1 hour. The washed microsomal pellets were resuspended in 50 mmollL TrislHCI buffer, pH 7.5, to a final concentration corresponding to 0.5 g liver equivalents per mL. Microsomal and cytosolic fractions were frozen in suitable aliquots in liquid nitrogen and stored at -80°C. Protein contents of liver microsomal fractions were determined according to Smith et al. (1985)

Microsomal incubations
The N-hydroxylation of the test article P5367 as well as of the reference compound MOCA was investigated in liver microsomal fractions from guinea pigs in the presence of an NADPH regenerating system. The incubations typically consisted of liver microsomal fractions (diluted to yield a final protein concentration in the range of about 0.5 to 4 mg/mL) in 0.1 mol/L TrislHCI buffer, pH 7.4, in a total volume of 1100 uL. An NADPH-regenerating system was used, containing final concentrations of 1 U/L of glucose-6-phosphate-dehydrogenase (Boehringer, Mannheim No. 127663), 1 mmol/L of NADP (Boehringer No. 128058), 5 mmol/L of glucose-6-phosphate (Boehringer, No. 127647) and 5 mmol/L ofMgCb. The test article P5367 as well as the reference compound MOCA were added as solutions in methanol to receive final concentrations of 2.5 to 100 JlmollL (final methanol content of the incubation mixture: 1% v/v). The incubations were performed at 37°C in a water bath for incubation times between 0 and 120 min. The incubations were stopped by two extractions of the parent compound and the N-hydroxylamines with one volume of methylene chloride. The methylene chloride extract was evaporated to dryness on a SpeedVac, the residue was dissolved in 50 flL acetonitrile and 40 flL were used for quantificatioon by reversed phase HPLC with UV detection. Additionally, 10 flL of the reconstituted extract were analysed by LC/MS. The recoveries for the parent compounds and its N-hydroxylamines and the limit of quantification for the NOH-P5367 were determined by analyses of microsomal incubations immediately after addition of known amounts of the investigated compounds. Yields of 100 and 93% (P5367, n=2), 95 and 89% (NOH-P5367, n=2), 77±1O% (MOCA, n=4) and 66±8% (NOH-MOCA, n=4) were obtained. The limit of quantification for NOH-P5367 was determined to be about 0.2 flmollL. The same limit of quantification was assumed for NOHMOCA.

Following incubation of both substances under optimal conditions (100 umol/litre substrate, 2mg/ml microsomal protein), formation of the N-hydroxylamine metabolite from the test substance P5367 was shown to occur.  The maximal amount of N-hydroxylamine formed was about 5-fold less than the equivalent metabolite produced for the reference
substance MbOCA.  The formation of the N-hydroxylamine metabolite was proportional with time up to 10 minutes incubation for both substances.  In the case of P-5367, at 15 minutes, N-hydroxylamine formation reached a plateau at around 5umol/litre which was maintained for a further 15 mins.  This was then followed by a rapid decline over the remaining 90 minutes of the experiment such that at 120 minutes the concentration of NOH-P5367 was about 0.45 uml/l. In marked contrast to P-5367, the concentration of NOH-MbOCA continued to increase up to 30 mins to a level of around 20umol/l at 120 minutes (i.e. essentially steady state was obtained between 30 and 120 minutes).

The formation of the N-hydroxylamine metabolites for both substances was proportional to the concentration of microsomal protein over a 15-minute incubation time up to 1 mg/ml of protein but was found to plateau with increasing protein concentration.  The initial N-hydroxylamine rate over 10 minutes was linearly dependent upon the substrate concentration for both substances, with formation of N-hydroxylamine metabolite considerably greater at lower substrate concentrations for MbOCA than for P5367.  So, for up to 50umol/l of parent substrate about 3-6% of P-5367 was found to convert to NOH-P5367 whereas 30-36% of MbOCA was converted at these substrate concentrations. Overall, therefore, the formation of NOH-P5367 under optimal conditions in this assay was significantly lower than the formation of NOH-MbOCA and this difference was found to be even greater at substrate concentrations of 50umol/l or less.  Under optimal conditions although NOH-P5367 was formed over the first 30 minutes, levels declined markedly over to subsequent 90 suggesting either more rapid conversion to a further metabolite and/or inactivation of P450.

Conclusions:

The data suggest that less N-hydroxylamine is metabolically available from P5367 than from MOCA indicating a lower genotoxic potential ofP5367. Indeed, in contrast to MOCA, P5367 was negative in vivo as well as in vitro in various short term tests for genotoxicity.

Description of key information

Toxicokinetic assessment

 

Physico-chemical properties 

The registered substance is an organic intermediate used in technical applications. The material consists of off-white crystals at room temperature, with a melting point of about 88°C and a boiling point in excess of 170°C which cannot however be determined because of decomposition. It has a molecular weight of 380 and is typically > 97.0% pure. The test item has a water solubility of 0.02 mg/L at 20°C, a fat solubility of 17000 mg/100 g standard fat HB307 and a partition coefficient of 6.3 (logPow). The vapour pressure is < 0.1 Pa at 25°C, the particle size determination indicates a fraction of about 8.5% with an aerodynamic diameter of less than 10 µm and about 1.1% with less than 2.0 µm.    

 

Toxicity 

 

Acute oral toxicity: 

The LD50 of the test substance was determined as > 5000 mg/kg using groups of five males and five female rats. One animal only showed clinical symptoms such as emaciation, hunched posture, stained fur and died on day 8. All other animals survived the treatment and were free of symptoms. No gross pathological changes were found in the survivors, the dead animal presented with dark lungs and pale, enlarged liver with dark and yellow patches. No mortality or clinical signs were observed in cats (five males and five females) after a single oral dose of 4000 mg/kg. 

Acute dermal toxicity: 

 

At a limit dose of 2000 mg/kg, no deaths occurred in rats. There were neither signs of local toxicity nor clinical signs of systemic toxicity. Lower than expected body weight gains were observed particularly in females. However, in absence of concurrent control values a relation to the treatment remains uncertain. 

 

Subacute (14 day) oral dose range finding study: 

 

Groups of three male and female rats were treated with 0, 1000, 3000 and 10000 ppm test substance in the diet. Three animals died at the high dose after 6, 12 and 15 days, respectively. Major signs of toxicity were emaciation, bodyweight loss and lethargy. Liver (males only) and adrenal weights were increased in all groups. Pathological changes were seen in high dose animals only and were haemorrhage of the stomach, bloody content of the urinary bladder and dilation of the urethra and/or renal pelvis. 

 

Subacute (28 day) oral toxicity: 

 

Groups of five male and five female rats were treated with 0, 100, 300 or 1000 ppm test substance in the diet. Bodyweight development, feed consumption and feed conversion efficiency were not affected by the treatment. Neither clinical signs nor mortality were observed. Treatment related findings were confined to the high dose and included liver weight increase and hypertrophy of centrilobular hepatocytes. The centrilobular hypertrophy was not accompanied by biochemical evidence of liver damage. No gross lesions were found at terminal necropsy. The no observed effect level (NOEL) was 300 ppm (corresponding to about 38 mg/kg/day). The high dose level of 1000 ppm (about 138.5 mg/kg/day) was generally well tolerated. 

 

Metabolism 

 

Mutagenicity - Metabolism:  

 

No indication for mutagenic potential was obtained in a battery of in vitro and in vivot ests. No toxicity was seen in the Ames test both in presence and absence of a rat liver post mitochondrial fraction (S-9 mix). In the in vitro cytogenetics assay with human lymphocytes, the test substance was apparently less toxic in presence of S-9 mix than in its absence.  

 

Assessment 

 

Although of moderately low molecular weight, the test substance will be physically available for absorption only to a limited extent because at room and body temperature, it is a solid with a very limited water solubility. The fat solubility and the high partition coefficient indicate a potential for bioaccumulation. 

 

The results of the oral studies indicate that the test substance is absorbed from the gastro-intestinal tract leading to clinical signs and, at high doses, to mortality. Adaptive liver changes and pathological lesions in the urinary tract observed in the subacute studies indicate that liver metabolism and renal excretion of the parent compound and/or metabolites occur. This is in agreement with the known metabolic and excretion pathways of other aromatic amines in rats.

 

The results of the acute dermal study did not reveal systemic toxicity. However, due to questionable findings on body weight gains the absorption by this route cannot ultimately be excluded. Inhalation of vapour at room temperature will be negligible based on the low vapour pressure.

 

Although the particle size distribution indicates a limited potential for inhalation, the absorption by the inhalation route is expected to be low based on the low water solubility and the deposition of the particles largely in the nasopharyngeal and tracheobronchial regions from where they may be cleared mechanical retrograde transport mechanisms. 

Additional information

Source: GLP-studies