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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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
distribution
excretion
Principles of method if other than guideline:
9 male Fischer rats were exposed intravenously to 2 mg/kg labeled 14C-MDA. Excreta and urine was collected after 6, 12, 24, 48, 72, and 96 h. After various time points rats were sacrificed and tissue distributon was determined.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and of test material: Aldrich Chemical Company

RADIOLABELLING INFORMATION
- Radiochemical purity: >99% (analysed by HPLC and TLC)
- Specific activity: 14.8 mCi/mmol
- Locations of the label: Ring-labeled 14C-methylenedianiline was prepared by the radiochemical synthesis section of MRI.

OTHER
The compound was received as the free base (3.2 mCi) in 100% ethanol or as the hydrochloride (8.18 mCi).
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
- Adult rats were purchased from Charles River Laboratories, North Wilmington, Massachusetts.
- Housing: 10-15 air changes per hour in polycarbonate cages
- Temperature/humidity: 72°C ±2/50% ±10
- 12h-Light/dark cycle
- Acclimatisation time: 7 days
- Diet: Purina Certified Rodent Chow (Ralston Purina Company)
- Drinking water ad libitum
- Weight: 149-218 g
Route of administration:
intravenous
Vehicle:
other: ethanol/water (1:4)
Duration and frequency of treatment / exposure:
single dose
Remarks:
Doses / Concentrations:
2 mg/kg bw (0.4 ml per animal)
No. of animals per sex per dose:
9 male rats
Control animals:
no
Details on study design:
Rats were treated and placed in individual metabolism cages.
Details on dosing and sampling:
Dosing was performed through the saphenous vein.
Excreta was collected after 6, 12, 24, 48, 72, and 96 h following administration. Three animals were sacrificed at 6, 24, and 96 h for tissue sampling. Urine was collected in containers kept on dry ice. After collection cages were rinsed and cage washings were analyzed. The animals were sacrificed and blood, tissue and organs were removed. Tissue and organs were washed with saline, blotted with absorbing paper, weighed and prepared for radiochemical analysis. All samples were kept on dry ice during necropsy.
Preliminary studies:
In a preliminary study (data not shown) the appropriate sampling times were selected and the methods of treatment and sampling determined.
In 3 rats most (approx. 95%) of the i.v. dose was eliminated in urine and feces during a 96-h period following dosing.
Details on distribution in tissues:
Significant amounts of radioactivity (24% at 6h) were recovered in the GI tract, probably through the bile. Blood and tissue contained 19% of the doses at 6h, but their 14C contents declined to 2% of the doses at 96h. The highest 14C-concentrations were demonstrated in the liver (9.5%, 4-9 times the blood level).
Details on excretion:
Total recoveries in excreta and tissue averaged 98.3, 98.1, and 99.3% at 6, 24, and 96 h after dosing.
During 96 h following i.v. dosing, 67.0 and 30.7% of doses were eliminated in urine and feces, respectively.
Metabolites identified:
not specified
Details on metabolites:
No data

Table 1: Recovery of radioactivity in rats treated i.v. with 4,4´-MDA.

recovery

Percent of dose

dose

0.4 mg/rat, i.v

time

6h

24h

96h

 blood

 2.8

0.44 

0.22 

 tissue

 16.6

4.33 

1.31 

 GI tract

 23.94

4.14 

0.11 

 urine

 54.65

67.35 

66.96 

 feces

 0.28

21.79 

30.66 

 total absorbed

 98.27

98.06 

99.25 

 dose wash

 -

 application area

 -

 recovery

 98.27

98.06 

99.25 

Table 2: Radioactivity in blood, tissue, and excreta of rats at 6 and 96 h following intravenous dose (0.4 mg) of 14C-MDA.

 n=3  % of dose (6h) % of dose (96h)
 Blood 2.8  0.22 
 Plasma 1.97  0.10 
 RBCs 0.55  0.09 
 Liver 9.47  0.86 
 Kidney 0.48  0.03 
 Lungs 0.26  0.02 
 Brain 0.08  0.00 
 Spleen 0.09  0.02 
 Testes 0.26  0.00 
 Adrenals 0.01  0.00 
 Bladder 0.00  0.00 
 Muscle 2.77  0.21 
Fat -
GI tract 23.94  0.11 
 Nontreated skin 3.19  0.16 
 Urine 54.65 66.96 
 Feces 0.28 30.66 
 Dose wash
 Application area
 Recovery 98.27  99.25 
 
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
distribution
excretion
Principles of method if other than guideline:
21 male Fisher rats were exposed dermally to 2 and 20 mg/kg labeled 14C-MDA. Excreta and urine was collected after 6, 12, 24, 48, 72, and 96 h. After various time points rats were sacrificed and tissue distributon was determined.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and of test material: Aldrich Chemical Company

RADIOLABELLING INFORMATION
- Radiochemical purity: >99% (analysed by HPLC and TLC)
- Specific activity: 14.8 mCi/mmol
- Locations of the label: Ring-labeled 14C-methylenedianiline was prepared by the radiochemical synthesis section of MRI.

OTHER
The compound was received as the free base (3.2 mCi) in 100% ethanol or as the hydrochloride (8.18 mCi).
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
- Adult rats were purchased from Charles River Laboratories, North Wilmington, Massachusetts.
- Housing: 10-15 air changes per hour.
- Temperature/humidity: 72°C ± 2/50% ± 10
- 12h-Light/dark cycle
- Acclimatisation time: 7 days
- Diet: Purina Certified Rodent Chow (Ralston Purina Company)
- Drinking water ad libitum
- Weight: 149-218 g
Route of administration:
dermal
Vehicle:
other: ethanol/water (95:5)
Details on exposure:
Labeled 14C-MDA and unlabeled MDA mixtures were dissolved in a ethanol/water (95:5) solution, and were applied at a volume of 0.1 ml/rat. The backs of the rats were lightly shaven with electric clippers before dosing. The substance was applied with a disposable micropipette on 2 cm2. The areas were normally covered with a wax-lined cup held in place by adhesive tape.
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
2 mg/kg bw (total dose)
Dose / conc.:
20 mg/kg bw (total dose)
No. of animals per sex per dose:
2 mg/kg: 15 rats
20 mg/kg: 6 rats
Control animals:
no
Details on study design:
2 mg/kg:
3 rats: After dosing rats were placed in a metabolism cage. At 6 h the application areas were washed with soap and water, and rats were sacrificed for skin and tissue sampling.
3 rats: After dosing rats were placed in a metabolism cage. At 24 h the application areas were washed with soap and water, and rats were sacrificed for skin and tissue sampling.
3 rats: After dosing rats were placed in a metabolism cage. At 96 h the application areas were washed with soap and water, and rats were sacrificed for skin and tissue sampling.
3 rats: After dosing rats were placed in a metabolism cage. At 24 h the application areas were washed with soap and water, and rats were placed again in the metabolism cage. At 96 h rats were sacrificed for skin and tissue sampling.
3 rats: After dosing (non-occlusive) rats were placed in a metabolism cage. At 6 h the application areas were washed with soap and water, and rats were sacrificed for skin and tissue sampling.

20 mg/kg:
3 rats: After dosing rats were placed in a metabolism cage. At 24 h the application areas were washed with soap and water, and rats were placed again in the metabolism cage. At 96 h rats were sacrificed for skin and tissue sampling.
3 rats: After dosing rats were placed in a metabolism cage. At 96 h the application areas were washed with soap and water, and rats were sacrificed for skin and tissue sampling.
Details on dosing and sampling:
Excreta was collected after 6, 12, 24, 48, 72, and 96 h following administration. Urine was collected in containers kept on dry ice.
After collection cages were rinsed and cage washings were analyzed.
The animals were sacrificed and blood, tissue and organs were removed. Tissue and organs were washed with saline, blotted with absorbing paper, weighed and prepared for radiochemical analysis. All samples were kept on dry ice during necropsy.
Preliminary studies:
In a preliminarily study (data not shown) the dermal washing efficiencies were studied to assess the extent of removal of the applied 14C-labeled MDA (2 mg/kg) by washing with soap or acetone solutions.
In 3 rats, higher and more consistent recoveries were obtained by washing the application areas with soap and water (91.3%) than with acetone and water (85.1%). Both soap and water or acetone and water were incapable of removing all the applied material. In addition, lower amounts were recovered in excreta when soap and water were used (1.6% after soap and water versus 7.7% after acetone and water). Recoveries of radioactivity from the application areas were lower after washing with soap and water (3.8%) than after acetone and water (12.7%).

In a second preliminary study (data not shown) the appropriate sampling times were selected and the methods of treatment and sampling determined. After dermal application of 2mg/kg to 3 rats, approx. 43% was eliminated in urine and feces in 96 h.
After dermal application of 20mg/kg to 3 rats, approx. 15% of the dose was recovered in excreta, suggesting limited absorption and/or elimination.
Details on absorption:
Absorbtion (Low dose; recovered in excreta and tissue): 11.9%, 27.8%, and 54.5% after 6, 24, and 96 h.
Absorbtion (Low dose; recovered in excreta, tissue, and application area): 42.5%, 53.6%, and 80.1%.
Absorbtion (Low dose; recovered in excreta and tissue; non-occluded): 7%
Absorbtion (Low dose; recovered in excreta, tissue, and application area; non-occluded): 34.5%
Absorbtion (Low dose; recovered in excreta and tissue; dose wash after 24 h): 42.9% after 96 h
Absorbtion (Low dose; recovered in excreta, tissue, and application area; dose wash after 24 h): 53% after 96 h
Absorbtion (High dose; recovered in excreta and tissue after 96 h): 6.6% (without dose wash at 24 h); 5.3% (with dose wash at 24 h).
Absorbtion (High dose; recovered in excreta, tissue, and application area): 30.6% (without dose wash at 24 h); 40.2% (with dose wash at 24 h).
Details on distribution in tissues:
Low dose: 3.8% and 4.7% were recovered in the GI tract and tissues, respectively, at 6h. The liver showed highest concentration (2%).
Higher tissue concentrations were demonstrated following the high dose of MDA, although the proportion of dose recovered in tissues was lower than found after the low dose. The distribution in tissues was similar after the low and high doses.
In animals treated dermally with the doses removed 24 h later, a rapid decline in tissue concentrations was demonstrated during the 72h recovery period, although liver-to-blood ratios remained high (data not shown).
Details on excretion:
At 6 h after treatment with low dose, 11.9% of the applied radioactivity was recovered in urine, GI tract and tissues. 62.1% and 30.5% were present in the dose wash and the application areas, respectively.
At 24 h after treatment with low dose, 27.8% of the applied radioactivity was recovered in feces, urine, GI tract and tissues. 52.1% and 25.8% were present in the dose wash and the application areas, respectively.
At 96 h after treatment with low dose, 54.7% of the applied radioactivity was recovered in feces, urine, GI tract and tissues. 24.7% and 25.6% were present in the dose wash and the application areas, respectively.
Rats in which the application areas were kept non-occluded showed lower amounts of 14C in the excreta, tissues, and the GI tracts (7% of the dose in 6 h). The remainder of the applied doses were recovered in the dose wash (70.2%) or from the application areas (27.5%). (data not shown)
In rats treated dermally with the low dose and the application areas washed at 24 h before the animals were returned to the metabolism cages, 42.9% of the doses were recovered in urine, feces, tissues, and GI tracts. 52.1% were recovered in the dose wash at 24 h. In addition, an average of 10.7% was recovered from the application areas at 96 h.
In animals treated with the high dose which was kept on the skin for 96 h, only 6.6% of the applied dose were recovered in urine, feces, GI tracts, and tissues. An average of 62.5% of the doses were washable and 24% recovered from the application areas. In the rats treated with the high dose (dose wash after 24 h) similar amounts (5.3%) were recovered in excreta and tissues during the 96-h collection period.
Metabolites identified:
not measured

Table 1: Recovery of radioactivity in rats treated dermal with 4,4´-MDA.

 

                   Percent of dose

         0.4 mg/rat  4 mg/rat
 

       dermal

  6 h  24 h  96 h  96 h 
 blood 0.77  0.29  0.11  0.03 
 tissue 4.7 2.15  0.88  0.22 
 GI tract 3.8  2.98  0.48  0.18 
 urine 2.55  20.03 43.04  4.82 
 feces 0.04  2.31  9.96  1.34 
 total absorbed 11.85  27.76  54.47  6.58 
 dose wash 62.08  52.08  24.67  62.45 
 application area 30.59  25.84 25.55  24.00 
 recovery 104.51  105.68  104.68  93.02 
         

Table 2: Radioactivity in blood, tissue, and excreta of rats at 96 h following dermal dose of 14C-MDA.

       % of dose  (6h)   % of dose  (96h)     
   0.4 mg dermal  4 mg dermal 0.4 mg dermal  4 mg dermal 
 Blood  0.77 0.08  0.11  0.03 
 Plasma  0.54 0.10  0.05  0.02 
 RBCs  0.17 0.05  0.04  0.01 
 Liver  1.967 0.53  0.54  0.11 
 Kidney  0.15 0.19  0.03  0.01 
 Lungs  0.03 0.05  0.00  0.00 
 Brain  0.03 0.01  0.00  0.00 
 Spleen  0.01 0.03 0.00  0.00 
 Testes  0.07 0.04 0.00  0.00 
 Adrenals  0.00 0.15 0.00  0.00 
 Bladder  0.00 0.00 0.00 0.00
 Muscle  1.191 0.02  0.10 0.04 
Fat  - 0.01 
GI tract  3.8 0.35 0.48  0.18 
 Nontreated skin  1.24 0.07  0.20  0.06 
 Urine  2.55- 43.04  4.82 
 Feces  0.04 9.96 1.34 
 Dose wash  62.08 24.67 62.45 
 Application area  30.59 25.55 24.00 
 Recovery  104.54 104.68 93.02 

Table 3: Percutaneous absorbtion of MDA based on recoveries in excreta, tissue, and skin.

exposure [h]

concentration [mg/rat]

% in excreta & tissue

abs. rate

[µg/h]

% skin appl. area

total

6

0.4

11.85

7.92

30.59

42.44

24

0.4

27.76

4.64

25.84

53.6

96

0.4

54.47

2.28

25.55

80.02

24/96

0.4

42.93

1.8

10.67

53.59

6h nonoccl.

0.4

7

4.68

27.53

34.53

96

4

6.58

2.8

24

30.58

24/96

4

5.27

2

8.93

14.2

 24/96h - dose was removed 24h following application and animals assayed after 96h

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
excretion
Principles of method if other than guideline:
2 monkeys (1 retested) were treated with 2 mg/kg MDA and transferred into metabolic chairs. Urine and feces were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h. One monkey was redosed 6 weeks later to obtain additional metabolism data.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and of test material: Aldrich Chemical Company

RADIOLABELLING INFORMATION
- Radiochemical purity: >99% (analysed by HPLC and TLC)
- Specific activity: 14.8 mCi/mmol
- Locations of the label: Ring-labeled 14C-methylenedianiline was prepared by the radiochemical synthesis section of MRI.

OTHER
The compound was received as the free base (3.2 mCi) in 100% ethanol or as the hydrochloride (8.18 mCi).
Radiolabelling:
yes
Remarks:
14C
Species:
monkey
Strain:
other: Rhesus
Sex:
male
Details on test animals and environmental conditions:
Adult monkeys were purchased from Hazleton Research Animals, Alice, Texas.
Housing: 10-15 air changes per hour in stainless steel cages
Temperature/humidity: 72°C ± 2 /50% ± 10
12h-Light/dark cycle
Acclimatisation time: 1 month
Diet: Purina Certified Primate Chow, No 5048. Supplemented with apples.
Route of administration:
intravenous
Vehicle:
other: Ethanol/Water (1:4)
Details on exposure:
A mixture of radiolabeled and normal MDA in ethanol/water was prepared.
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
2 mg/kg bw (total dose)
Remarks:
5 mL of a MDA-solution in ethanol/water
No. of animals per sex per dose:
2 male monkeys (1 retested)
Control animals:
not specified
Details on study design:
2 monkeys were treated with 2 mg/kg MDA and transferred into metabolic chairs. Urine and feces were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h. One monkey was redosed 6 weeks later to obtain additional metabolism data.
Details on dosing and sampling:
Dosing was performed through the saphenous vein.
Excreta was collected after 6, 12, 24, 48, 72, 96, 120, 144, and 168 h following administration.
Details on distribution in tissues:
not assayed
Details on excretion:
Almost complete recovery (93-98%) was obtained from the monkeys.
Excretion of 14C-MDA occured primarily in the urine (84.3%) and to a lesser extent in the feces (9.8%). Peak elimination occured at 6 to 12 h, although 1 animal showed considerable 14C-elimination between 24 and 48 h.
Metabolites identified:
not measured
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
excretion
Principles of method if other than guideline:
2 monkeys (1 retested after 6 weeks) were treated dermally with 2 mg/kg MDA and transferred into metabolic chairs for 24 h. At 24 h application site was washed and monkeys were transferred to individual metabolism cages. Urine and feces were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and of test material: Aldrich Chemical Company

RADIOLABELLING INFORMATION
- Radiochemical purity: >99% (analysed by HPLC and TLC)
- Specific activity: 14.8 mCi/mmol
- Locations of the label: Ring-labeled 14C-methylenedianiline was prepared by the radiochemical synthesis section of MRI.

OTHER
The compound was received as the free base (3.2 mCi) in 100% ethanol or as the hydrochloride (8.18 mCi).
Radiolabelling:
yes
Remarks:
14C
Species:
monkey
Strain:
other: rhesus
Sex:
male
Details on test animals and environmental conditions:
Adult monkeys were purchased from Hazleton Research Animals, Alice, Texas.
Housing: 10-15 air changes per hour in stainless steel cages
Temperature/humidity: 72°C ± 2 /50% ± 10
12h-Light/dark cycle
Acclimatisation time: 1 month
Diet: Purina Certified Primate Chow, No 5048. Supplemented with apples.
Route of administration:
dermal
Vehicle:
other: Ethanol/Water (95:5)
Details on exposure:
A mixture of radiolabeled and normal MDA in ethanol/water was prepared.
The lateral forearms of the monkeys were lightly shaven with electric clippers shortly before dosing. 1.25 ml per animal was applied with a disposable micropipette on a specific area 50 cm². Except when indicated otherwise, the dosed areas were covered with a wax-lined cup held in place by adhesive tape.
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
2 mg/kg bw (total dose)
No. of animals per sex per dose:
2 male monkeys (1 retested)
Control animals:
no
Details on study design:
2 monkeys were treated with 2 mg/kg MDA and transferred into metabolic chairs for the first 24 h. The dermal application areas were then washed with soap and water and the monkeys were transferred to individual metabolism cages for the remainder of the study.
Urine and feces were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h. One monkey was redosed 6 weeks later to obtain additional metabolism data.
The monkeys were not sacrificed.
Cage washings were analysed for ratioactivity.
Details on dosing and sampling:
Excreta was collected after 6, 12, 24, 48, 72, 96, 120, 144, and 168 h following administration.
Details on absorption:
Based on the excretory data only, the amounts considered to be absorbed through the skin averaged 20.8% of the dose. Since the application area was not sampled the total amount absorbed was estimated to be 52.8% if quantitative recovery (100%) is achievable.
Details on distribution in tissues:
no data
Details on excretion:
47.3% of the dose was recovered in the washing solution.
18.8% and 1.4% were recovered in the urine and feces during the 7-day collection period, respectively.
Excretion peaked at 24 h for 1 monkey and at 48 h for the other two monkeys.
Total recoveries in skin wash and excreta averaged only 68%.
Metabolites identified:
not measured

Table 1: Percutaneous absorbtion of MDA based on recoveries in excreta, tissue, and skin.

exposure [h]

concentration [mg/animal]

% in excreta & urine

abs. rate

[% dose/h]

24/96

10

18.04

18

24/168

10

20.76

12

expousue for 24h followed by washing, sacrificition after 96 or 168h.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Principles of method if other than guideline:
Investigation of percutaneous absorption of 4.4'-methylenedianiline (MDA) after topical application to full-thickness skin from male Fischer 344 rats in flow-through diffusion cells, on four separate occasions.
GLP compliance:
no
Specific details on test material used for the study:
RADIOLABELLING INFORMATION
- Radiochemical purity: >95%
- Specific activity: 56 mCi/mmol
- Locations of the label: [ring14C]4,4 '-methylenedianiline (Amersham)
Radiolabelling:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-OLAC (Oxford, Oxon, UK)
- Diet: CRM pellets, Biosure (Manea, Cambs., UK); ad libitum
- Water: ad libitum
Type of coverage:
other: occlusive and open
Vehicle:
ethanol
Duration of exposure:
72 h
Doses:
- Actual doses: 39.4, 20.1, 21.6, 23.6 µg/cm²
- Dose volume: 5-10 µL
No. of animals per group:
4
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: dorsal region of male Fischer 344 rats
- Type of skin: full thickness
- Preparative technique: following sacrificion, the dorsal region was shaved with animal clippers and the shaved area was cut out with dissecting scissors. The rat skin sections were placed, epidermal side uppermost, on a plastic dissecting board and circles, 1.7 cm in diameter, were cut out using a circular sharpened steel cutter. Excess subcutaneous tissue was removed with a scalpel.

PRINCIPLES OF ASSAY
- Flow-through diffusion cell system: consisted of seven teflon diffusion cells and a fraction collector (Crown Glass Co Inc., Somerville, NJ, USA), a thermostatically controlled Churchill water circulator and a model 202V/AA16 cassette peristaltic pump (Watson-Marlow, Falmouth, Cornwall, UK). The receptor volume of each diffusion cell was 130 ul.
- Receptor fluid: HEPES buffered Hanks' balanced salt solution supplemented with 0.5% (v/v) gentamicin, continuously flowing
- Test temperature: 32°C
- the skin was placed in the diffusion cells in the flow-through apparatus, and allowed to equilibrate for about 30min.
- aliquots of 5-10 µL [14C]-MDA (17.7-40.6 µg/cm²) in ethanol were applied to the exposed skin surface.
- occlusion with teflon caps or left open.
- receptor fluid (1.5 mL) was collected hourly for up to 72h.
- at the end of the experiment (72h) the epidermal surface of the skin was washed, and the washings and skin were counted to determine radioactivity remaining on the skin surface.

ANALYSIS
- Liquid scintillation counting

STATISTICAL EVALUATION:
- Student`s t-Test for unpaired data, level of significance p<0.05.
Absorption in different matrices:
See table 1
Total recovery:
- Total recovery: 94.5 +/-6 %
Dose:
20.1-39.4 µg/cm³
Parameter:
percentage
Absorption:
ca. 6.1 %
Remarks on result:
other: 72 h
Remarks:
open
Dose:
20.1-39.4 µg/cm²
Parameter:
percentage
Absorption:
ca. 13.3 %
Remarks on result:
other: 72h
Remarks:
occlusive

Table 1: Recovery of radioactivity at 72h after topical application of [14C] MDA to rat skin in vitro (% of applied dose).

Rat (unoccluded) (n=12) Rat (occluded) (n=16)
Receptor fluid 6.1 ± 2 13.3 ± 4.0*
Skin surface 25.5 ± 6.8 21.5 ± 7.2
Diffusion cell 5.4 ± 4.4 3.9 ± 2.6
Teflon cap - 0.3 ± 0.2
Skin surface 57 ± 5.6 53.1 ± 8.6
Total 94.5 ± 6.0 92.1 ± 6.5

* significantly different from unoccluded value (p<0.05)

A typical absorption profile showed a lag phase of about 2 hr before a rapid increase in absorption, reaching an equilibrium at 72 hr (data not shown).

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Principles of method if other than guideline:
Investigation of percutaneous absorption 4.4'-methylenedianiline (MDA) after topical application to full-thickness skin from humans in flow-through diffusion cells, on four separate occasions.
GLP compliance:
no
Specific details on test material used for the study:
RADIOLABELLING INFORMATION
- Radiochemical purity: >95%
- Specific activity: 56 mCi/mmol
- Locations of the label: [ring14C]4,4 '-methylenedianiline (Amersham)
Radiolabelling:
yes
Species:
human
Sex:
not specified
Details on test animals and environmental conditions:
human skin, from surgical resection
Type of coverage:
other: occlusive and open
Vehicle:
ethanol
Duration of exposure:
72 h
Doses:
- Actual doses: 17.7; 17.7; 19.5; 40.6 µg/cm²
- Dose volume: 5-10 µL

No. of animals per group:
4
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human, from surgical resection
- Type of skin: Full-thickness, healthy human breast skin
- Preparative technique: trimmed of excess subcutaneous tissue with a scalpel

PRINCIPLES OF ASSAY
- Flow-through diffusion cell system: consisted of seven teflon diffusion cells and a fraction collector (Crown Glass Co Inc., Somerville, NJ, USA), a thermostatically controlled Churchill water circulator and a model 202V/AA16 cassette peristaltic pump (Watson-Marlow, Falmouth, Cornwall, UK). The receptor volume of each diffusion cell was 130 µL.
- Receptor fluid: HEPES buffered Hanks' balanced salt solution supplemented with 0.5% (v/v) gentamicin, continuously flowing
- Test temperature: 32°C
- the skin was placed in the diffusion cells in the flow-through apparatus, and allowed to equilibrate for about 30 min.
- aliquots of 5-10 µL [14C]-MDA (17.7-40.6 µg/cm²) in ethanol were applied to the exposed skin surface.
- occlusion with teflon caps or left open.
- receptor fluid (1.5 mL) was collected hourly for up to 72h.
- at the end of the experiment (72h) the epidermal surface of the skin was washed, and the washings and skin were counted to determine radioactivity remaining on the skin surface.

ANALYSIS
- Liquid scintillation counting

STATISTICAL EVALUATION:
- Student`s t-Test for unpaired data, level of significance p<0.05.
Total recovery:
88.7-93.4%
Dose:
17.7 - 40.6 μg/cm²
Parameter:
percentage
Absorption:
ca. 13 %
Remarks on result:
other: 72 h
Remarks:
unocclusive
Dose:
17.7 - 40.6 μg/cm²
Parameter:
percentage
Absorption:
ca. 33 %
Remarks on result:
other: 72h
Remarks:
occlusive
Conversion factor human vs. animal skin:
inter-individual coefficient of variation in the mean absorption MDA through unoccluded human skin was 33.1%, and this variation was unaffected by occlusion.

 Table 1: Recovery of radioactivity at 72 h after topical application of [14C] MDA to human skin in vitro (% of applied dose).

Human (unoccluded) (n=12) Human (occluded) (n=16)
Receptor fluid 13.0  ± 4.3 32.9 ± 9.0*
Skin surface 36.5 ± 10.7 17.3 ± 9.6*
Diffusion cell 15.4 ± 12.3 5.6 ± 5.6*
Teflon cap - 0.1 ± 0.1
Skin surface 23.8 ± 13.6 37.4 ± 10.3
Total 88.7 ± 5.4 93.4 ± 4.5

* Significantly different from corresponding unoccluded values (p<0.05).

A typical absorption profile showed a lag phase of about 2 hr before a rapid increase in absorption, reaching an equilibrium at 72hr (data not shown).

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Absorption

Following dermal application (2 mg/kg bw onto 2 cm² 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) (US EPA, 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/cm² were 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).

In a study performed by E.I. du Pont de Nemours & Co (1974), dermal absorption of MDA in dogs was investigated. No MDA was detected in blood, and only traces of MDA (although not in a dose-dependent manner) were found in urine. The study is considered not assignable due to insufficient documentation.

In an in vitro study, Hinz et al. (1991) investigated the percutaneous penetration of MDA through skin samples from hairless mice. Depending on the surface concentration applied and on the vehicle used, cumulative absorptions after 16 hours of 4.5 to 240

µg were found.

In an insufficiently documented percutaneous absorption study (Huntsman, 1969), the authors reported that 14C-MDA in THFA readily passes through the skin of rats, with about double the absorption in 24 hours compared with 4 hours.

 

Distribution

Following dermal and intravenous application in rats and guinea pigs (US EPA, 1986) and intraperitoneal application in rats and rabbits (Morgott, 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., 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 (US EPA, 1986), again the liver seems to be the major tissue for recovery of radioactivity.

 

Excretion

The fate of MDA seems to vary in 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, 1984). Since the compound was administered by the intraperitoneal route, the amount of fecal radioactivity provided an indication of biliary 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, significantly higher urinary excretion 96 hours following i.v. dosing (67% in urine and 30.7% in feces) was reported for rats compared to i.p. dosing in the study of Morgott (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 (US EPA, 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 96 hours 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 (US EPA, 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. 

In a mouse study the time course of elimination following a single intraperitoneal application of 14C-MDA was determined. The elimination rate was calculated as 0.22 h-1, resulting in a half-life (t1/2) of 3.2 h (Tortoreto et al., 1983). Unfortunately no raw data is provided to comprehensively reproduce calculations.

 

Metabolism

Although differences in the quantitative aspects of metabolism remain unsolved, 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 supposed 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 being 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 neither glucuronide nor 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.

 In vitro metabolism of MDA was investigated using rabbit liver microsomes (Kajbaf et al., 1992) in which only CYP-dependent metabolism can be observed. The following three metabolites were detected: azo-MDA, azoxy-MDA, and nitroso-MDA (4-nitroso-4'-aminodiphenylmethane). The azo- and azoxy-compounds were formed enzymatically, whereas the nitroso-compound may have been formed via a non-enzymatic process. The hydroxylamine of MDA was not detected in this study. However, its initial formation has to be supposed as prerequisite for the formation of the dimeric MDA metabolites azo- and azoxy-MDA.

Formica (1984) reported detection of MDA in the urine of rats orally treated with a single dose of 68.3 mg/kg bw (gavage), but the documentation is insufficient for assessment.

 

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 et 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.

Formation of DNA adducts was reported from livers of rats after i.p. application of radiolabeled MDA (Schütze et al. 1996).

In an insufficiently documented study, Farmer and Bailey (1989) reported the detection of sulphinic acid amide adducts of MDA with hemoglobin in rats. The amount of adduct derived from a rat exposed to 2 mg/kg MDA was 2.3 ng MDA/µmol hemoglobin.

Sagelsdorff and Buser (1995) treated rats with single i.p. doses of MDA (0.2, 2, 20 mg/kg bw) and investigated formation of DNA- and Hb-adducts in blood and liver at various time points. The calculated covalent binding index (CBI) was in the range of 1 -2, which is typically found with weak genotoxic carcinogens. The extent of hemoglobin-bound radioactivity correlated well with the DNA-bound radioactivity in individual animals.

Endo and Hara (1991) reported MDA adduct formation with rat liver DNA, but the documentation is insufficient for assessment.

Disregarded studies

BASF (1962) investigated the percutaneous resorption toxicity of MDA in rats and cats. The animals were dermally exposed to the test substance under occlusive dressing. Clinical signs were observed, indicating that the test substance was dermally absorbed.

However determination of the absorption rate was not possible because only urine (no feces, no blood) was analysed and no skin samples were taken. Furthermore, the control animals partly showed similar symptoms as the treated animals. Thus, the study is considered unreliable and not used for assessment.