2,2',2''-nitrilotriethanol

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: Basic data given acceptable for assessment

Principles of method if other than guideline:

Groups of four female mice received a single intravenous dose of 3 mg/kg [14C]-triethanolamine. Expired radioactivity was trapped and quantitated and urine and feces were collected from all B6C3F1 mice dosed intravenously up to 72 hours after dosing. Tissue samples at 72 hours after dosing were also examined.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

mouse

Strain:

B6C3F1

Sex:

female

Route of administration:

intravenous

Vehicle:

acetone

Details on exposure:

Single intravenous doses contained approximately 6 μCi radiolabel for mice, an appropriate amount of nonradiolabeled triethanolamine, and isotonic saline as a vehicle that delivered a total dosing volume of 2 mL/kg to mice. Intravenous doses were drawn into a syringe equipped with a Teflon®-tipped plunger (Hamilton) and a 30 gauge hypodermic needle. Excess dose formulation was wiped off the needle before weighing the filled dosing syringe. Intravenous doses were injected into one lateral tail vein. After dosing, the needle was wiped clean with a Kimwipe®, and the empty syringe was reweighed. The Kimwipe® was placed into a vial containing 2 mL ethanol and analyzed by liquid scintillation spectrometry. Each dose was calculated as the difference between the weights of the filled and empty dosing apparatus less the amount found in the Kimwipe®. To determine the concentration of [14C]-triethanolamine in the dose formulation, two weighed aliquots were taken before, two after, and one during dosing.

Duration and frequency of treatment / exposure:

72 hrs

Dose / conc.:

3 other: mg/kg bw

No. of animals per sex per dose / concentration:

4

Control animals:

no

Positive control reference chemical:

not done

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues, cage washes, CO2
- Time and frequency of sampling: 24, 48, 72 hrs

Details on distribution in tissues:

The distribution of radioactivity present in tissue samples from female mice showed that the heart, kidney, liver, lung, and spleen contained higher concentrations of triethanolamine equivalent relative to blood.

Details on excretion:

26% of the dose was recovered in the urine within 24 hours.
An average of 62% of the dose was recovered in the urine within 72 hours after dosing, and 27.6% was recovered in the feces during this time.

Details on metabolites:

Urine collected 6 to 24 hours after intravenous dosing contained more than 95% radiolabeled components that coeluted with unchanged triethanolamine, with minor components eluting the same fractions in mice as those in rats.

With only 40% of the dose excreted within 24 hours, mice appeared to excrete intravenously administered triethanolamine much slower than did rats. Considerably more of the dose was recovered in the feces of mice (28 %) than in rats (0.6 %). However, it is common for mice to shred and powder their food pellets, and the feces collections are often contaminated with urine-soaked solids and their associated radiochemical equivalents. Less than 0.5 % of the dose was recovered in carbon dioxide traps, and less than 0.1 % was recovered in volatiles traps. The distribution of radioactivity present in tissue samples from female mice showed that, as with rats, the heart, kidney, liver, lung, and spleen contained higher concentrations of triethanolamine equivalent relative to blood.

Reference 2

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: Basic data given acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of four female mice received a single dermal dose of 79 or 1120 mg/kg bw. Expired radioactivity was trapped and quantitated and urine and feces were collected from all B6C3F1 mice dosed via skin painting up to 72 hours after dosing.The skin site of application from all mice was examined.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

mouse

Strain:

B6C3F1

Sex:

female

Type of coverage:

semiocclusive

Vehicle:

acetone

Duration of exposure:

72 hrs

Doses:

79 or 1120 mg/kg bw

No. of animals per group:

4

Control animals:

no

Details on study design:

Dermal doses were formulated in acetone and contained 65 μCi radiolabel for rats and 12 to 15 μCi for mice with an appropriate amount of nonradiolabeled triethanolamine in a volume of approximately 190 μL per dose. A dose area of 12 cm2 for rats and 1.44 cm2 for mice was located on each animal’s back. Approximately 24 hours prior to dosing, rats were anesthetized with 7:1 ketamine:xylazine (60 mg/kg) by intramuscular injection and mice with sodium pentobarbitol (60 mg/kg) by intraperitoneal injection. Fur at the dose area was clipped with a No. 40 blade (Oster Professional Products). The clipped area on each animal was wiped with a gauze soaked with acetone, dried, and then examined for abrasions. Any animal with abrasions in the clipped area was excluded from the study. The dose area was outlined on the animal’s back with a permanent marker, and the animal was placed in a metabolism cage.

The doses for mice were administered over the dose area using a 100 or 250 μL syringe equipped with a Teflon®-tipped plunger and a gavage needle. A metal tissue capsule was glued in place over the dose area using Duro Quick Gel™ Super Glue.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

79 mg/kg bw group
Blood: 0.00590%
Dose site: 1.35%
Faeces: 7.80%
Urine: 48.2%
Total: 57.3%
14.6% remained unabsorbed (dosing appliance, skin gauze, skin wash)

1120 mg/kg bw group
Blood: 0.00627%
Dose site: 0.576%
Faeces: 13.0%
Urine: 67.7%
Total: 81.3%
6.75% remained unabsorbed (dosing appliance, skin gauze, skin wash)

Total recovery:

60% to 80% of dermally applied 79 and 1120 mg/kg triethanolamine was absorbed by female mice within 72 hours.

On the basis of mass of triethanolamine per area of skin, the lowest dermal dose levels for rats and mice were equal at 1.09 mg/cm2. The skin of mice is thinner than that of rats, and this difference may explain the higher percentage of dose absorbed by mice. The highest dermal doses were 4 and 15 mg/cm2 for rats and mice, respectively. Triethanolamine enhances its own absorption, and the pronounced difference between the species was not unexpected. The percent of dose absorbed in each species increased with increasing dose, but in rats, the increase was not statistically significant. Both species rapidly excreted the absorbed dose, primarily in urine. In rats, less than 1% of the dose was present in the tissue samples (except the dose site) 72 hours after treatment; the heart, kidney, liver, lung, and spleen contained elevated concentrations of radiolabel relative to blood.

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: Basic data given acceptable for assessment

Principles of method if other than guideline:

Groups of four female rats received a single dermal dose of 68 or 276 mg/kg bw. Expired radioactivity was trapped and quantitated and urine and feces were collected from all F344/N rats dosed via skin painting up to 72 hours after dosing. Tissue samples from all dermal rats 72 hours after dosing were also examined.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Fischer 344

Sex:

female

Type of coverage:

semiocclusive

Vehicle:

acetone

Duration of exposure:

72 hrs

Doses:

68 or 276 mg/kg bw

No. of animals per group:

4

Control animals:

no

Details on study design:

Dermal doses were formulated in acetone and contained 65 μCi radiolabel for rats with an appropriate amount of nonradiolabeled triethanolamine in a volume of approximately 190 μL per dose. A dose area of 12 cm2 for rats was located on each animal’s back. Approximately 24 hours prior to dosing, rats were anesthetized with 7:1 ketamine:xylazine (60 mg/kg) by intramuscular injection. Fur at the dose area was clipped with a No. 40 blade (Oster Professional Products). The clipped area on each animal was wiped with a gauze soaked with acetone, dried, and then examined for abrasions. Any animal with abrasions in the clipped area was excluded from the study. The dose area was outlined on the animal’s back with a permanent marker, and the animal was placed in a metabolism cage.

Prior to dermal dosing, a protective foam appliance was glued to each rat’s back using Hollister’s Medical Adhesive. The doses were administered evenly over the dose area using a 500 μL syringe equipped with a Teflon®-tipped plunger and a gavage needle. A nonocclusive cloth cover was attached to the appliance, and a protective metal mesh cover was secured over the appliance with Elastoplast adhesive bandage prior to returning the rat to its cage.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

68 mg/kg bw group
Tissues: 0.688%
Dose site: 5.62%
Faeces: 0.247%
Urine: 12.9%
Total: 19.4%
66.5% remained unabsorbed (dosing appliance, skin gauze, skin wash)

276 mg/kg bw group
Tissues: 1.36%
Dose site: 3.12%
Faeces: 0.142%
Urine: 23.8%
Total: 28.4%
54.6% remained unabsorbed (dosing appliance, skin gauze, skin wash)

Total recovery:

Only 20% to 30% of dermally applied 68 and 276 mg/kg triethanolamine was absorbed by female rats within 72 hours.

Urine collected 48 to 72 hours after dermal application of triethanolamine in female rats was analyzed by HPLC. The chromatogram contains a peak that coelutes with triethanolamine and two other peaks that comprise about 5% of the radioactivity in the sample. These peaks, however, were also present in the chromatogram of the radiolabeled test article and may reflect the presence of impurities rather than metabolites. The metabolite fractions were collected and incubated with purified beta-glucuronidase, as was an aliquot of the whole urine. Analysis of these samples showed no change in the metabolite profile.

On the basis of mass of triethanolamine per area of skin, the lowest dermal dose levels for rats and mice were equal at 1.09 mg/cm2. The skin of mice is thinner than that of rats, and this difference may explain the higher percentage of dose absorbed by mice. The highest dermal doses were 4 and 15 mg/cm2 for rats and mice, respectively. Triethanolamine enhances its own absorption, and the pronounced difference between the species was not unexpected. The percent of dose absorbed in each species increased with increasing dose, but in rats, the increase was not statistically significant. Both species rapidly excreted the absorbed dose, primarily in urine. In rats, less than 1% of the dose was present in the tissue samples (except the dose site) 72 hours after treatment; the heart, kidney, liver, lung, and spleen contained elevated concentrations of radiolabel relative to blood.

Reference 4

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: Basic data given acceptable for assessment

Principles of method if other than guideline:

Groups of four female rats received a single intravenous dose of 3 mg/kg [14C]-triethanolamine. Expired radioactivity was trapped and quantitated and urine and feces were collected from all F344/N rats up to 72 hours after dosing. Tissue samples at 72 hours after dosing were also examined.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Fischer 344

Sex:

female

Route of administration:

intravenous

Details on exposure:

Single intravenous doses contained approximately 47 μCi radiolabel for rats, an appropriate amount of nonradiolabeled triethanolamine, and isotonic saline as a vehicle that delivered a total dosing volume of 1 mL/kg to rats. Intravenous doses were drawn into a syringe equipped with a Teflon®-tipped plunger (Hamilton) and a 27 gauge hypodermic needle. Excess dose formulation was wiped off the needle before weighing the filled dosing syringe. Intravenous doses were injected into one lateral tail vein. After dosing, the needle was wiped clean with a Kimwipe®, and the empty syringe was reweighed. The Kimwipe® was placed into a vial containing 2 mL ethanol and analyzed by liquid scintillation spectrometry. Each dose was calculated as the difference between the weights of the filled and empty dosing apparatus less the amount found in the Kimwipe®. To determine the concentration of [14C]-triethanolamine in the dose formulation, two weighed aliquots were taken before, two after, and one during dosing.

Duration and frequency of treatment / exposure:

72 hrs

Dose / conc.:

3 other: 3 mg/kg bw

Control animals:

no

Positive control reference chemical:

not done

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues, cage washes, CO2
- Time and frequency of sampling: 24, 48, 72 hrs

Details on absorption:

The radioactivity was rapidly excreted in the urine, and 90% of the dosed radioactivity was recovered in the urine within 24 hours. An average of 98% of the dose was recovered in the urine within 72 hour after dosing, and approximately 0.6% of the radioactivity was recovered in the feces during this time. Less than 0.5% of the dose was recovered in carbon dioxide traps, and less than 0.1% was recovered in volatiles traps.

Details on distribution in tissues:

0.9% of the dose remained in the tissues 72 hours after dosing

Details on excretion:

90% of the dosed radioactivity was recovered in the urine within 24 hours.
An average of 98% of the dose was recovered in the urine within 72 hour after dosing, and approximately 0.6% of the radioactivity was recovered in the feces during this time.

Metabolites identified:

yes

Details on metabolites:

Urine collected 6 to 24 hours after intravenous dosing and 48 to 72 hours after dermal application of triethanolamine in female rats was analyzed by HPLC. The chromatogram obtained contained a peak that coeluted with triethanolamine and two other peaks that comprise about 5% of the radioactivity in the sample. These peaks, however, were also present in the chromatogram of the radiolabeled test article and may reflect the presence of impurities rather than metabolites. The metabolite fractions were collected and incubated with purified beta-glucuronidase, as was an aliquot of the whole urine. Analysis of these samples showed no change in the metabolite profile

The distribution of radioactivity from female rats following intravenous administration indicated that only 0.9% of the dose remained in the tissues 72 hours after dosing. In contrast to diethanolamine, which was only slowly excreted (30% of dose within 48 hours) and accumulated in the brain, heart, kidney, spleen (5% of the dose at 48 hours), and liver (27% of dose at 48 hours) in a process thought to involve biochemical mimicry with the natural alkanolamine ethanolamine, comparatively little triethanolamine bioaccumulated in the tissues. Urine collected 6 to 24 hours after intravenous dosing and 48 to 72 hours after dermal application of triethanolamine in female rats was analyzed by HPLC. The chromatogram obtained contained a peak that coeluted with triethanolamine and two other peaks that comprise about 5% of the radioactivity in the sample. These peaks, however, were also present in the chromatogram of the radiolabeled test article itself and may reflect the presence of impurities rather than metabolites. The metabolite fractions were collected and incubated with purified beta-glucuronidase, as was an aliquot of the whole urine. Analysis of these samples showed no change in the metabolite profile.

Reference 5

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:

toxicokinetics

Principles of method if other than guideline:

The distribution and excretion of (14)C-TEA following intravenous application of 1 mg/kg bw was determined in male C3H/HeJ mice.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7H-5317

RADIOLABELLING INFORMATION
- Radiochemical purity: 98.6 %
- Specific activity: 34.7 mCi/mmol

Radiolabelling:

yes

Remarks:

14C

Species:

mouse

Strain:

other: C3H/HeJ

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Habour, ME, USA
- Weight at study initiation: 20.4 - 38.3 g
- Housing: 1-3/cage prior to study
- Individual metabolism cages: yes
- Diet: Purina Certified Rodent Chow 5002 ad libitum
- Water: tap water ad libitum):
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-60
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intravenous

Vehicle:

water

Details on exposure:

Intravenaus administration.
A aqueous solution of 14C-TEA with a concentration of approximately 0.5 mg/ml was administered intravenously via the tail vein to twenty-seven male mice. To facilitate making the injections, a device similar to that described by Keighley, 1966 was used for transillumination of the tail, and a device similar to that described by Grice, 1964 was used for restraining the mice. The dosing volume administered intravenously was 95.17 ± 6.79 % (mean, ± S.D.) of the target dosing volume of 2 ml/kg body weight. The target dose level was 1 mg TEA /kg body weight and the average amount of radioactivity injected per mouse was 4.69 µCi/mouse. The specific activity of the dosing solution was 30.7 µCi/g.
Immediately following dosing, the animals were returned to their cages, with the animals designated for excreta collection and sacrifice at 24 hours postdosing (final sacifice) placed in Roth cages. Blood samples were coIlected from all animals via orbital sinus puncture just prior to their sacrifice by cervical dislocation in groups of three at 0.083, 0.167, 0.5, 1, 2, 4, 6, 12 and 24 hours post-dosing.
Urine, faeces and expired C02 were collected at 12 and 24 hours from the mice in Roth cages. Urine and feces were collected in containers cooled on dry ice. Expired C02 was trapped in a solution of monoethanolamine: ethylene glycol monornethyl ether (3:7). Subsequent experiments did not include trapping of expired C02 since none of administered dose was excreted via this route following intravenous administration. Following the sacrifice of this group, the Roth cage was washed with a solution of acetone and water. Radioactivity in the excreta, CO2 trapping solutions, cage wash solutions, liver, kidneys, skin and remaining carcass were determined.

Duration and frequency of treatment / exposure:

single treatment

Dose / conc.:

1 other: mg/kg bw

No. of animals per sex per dose / concentration:

27

Control animals:

no

Positive control reference chemical:

None

Details on study design:

None

Details on dosing and sampling:

Immediately following dosing, the animals were returned to their cages, with the animals designated for excreta collection and sacrifice at 24 hours post-dosing final sacrifice) placed in Roth cages. Blood samples were collected from all animals via orbital sinus puncture just prior to their sacrifice by cervical dislocation in groups of three at 0.083, 0.167, 0.5, 1, 2, 4, 6, 12 and 24 hours post-dosing.
Urine, feces and expired CO2 were collected at 12 and 24 hours from the mice in Roth cages. Urine and feces were collected in containers cooled on dry ice.
Expired CO2 was trapped in a solution of monoethanolamine:ethylene glycol monomethyl ether (3:7). Subsequent experiments did not include trapping expired CO2 since none of administered dose was excreted via this route following intravenous administration. Following the sacrifice of this group, the tails were removed, the animals were skinned, and the liver and kidneys excised. Following sacrifice, each Roth cage was washed with a solution of acetone and water. Radioactivity in the excreta, CO2 trapping solutions, cage wash solutions, liver, kidneys, skin and remaining carcass were determined.

Preliminary studies:

Not applicable

Details on distribution in tissues:

The average amount of the administered dose remaining in the body at sacrifice was 3.6% of which 1% or less was found in the liver, kidneys and skin.

Details on excretion:

Urine was the primary route for elimination of radioactivity following intravenous administration of 14C-TEA. Feces was the secondary route of elimination for the route under investigation.

Metabolites identified:

not measured

Blood concentration-time profile in mice following intravenous administration:

Following a 1 mg/kg intravenous dose of 14C-TEA, the radioactivity in the blood was eliminated from the blood by an apparent firstorder process in a biphasic manner. The half-life of the alpha-phase of elimination of radioactivity from the blood was 0.58 hours with a terminal half-life for the elimination of radioactivity of 10.2 hours as estimated by the method of residuals (Gibaldi and Perrier, 1982).

Recovery of the administered dose following intravenous administration to mice:

Urine was the primary route for elimination of radioactivity following intravenous administration of 14C-TEA. Approximately 65% of the administered dose was excreted via this route of elimination within 24 hours post-dosing. Feces was the secondary route of elimination for the route under investigation. The mean percentage eliminated via the feces ranged from 16.3 to 27.7. There were no marked differences observed across dose levels or routes of administration in the amounts of the dose eliminated via the urine and feces. The average amount of the dose remaining in the body at sacrifice was 3.6% for intravenously dosed animals. The average amount of the administered dose found in the liver, kidneys and skin was approximately 1% or less.

Reference 6

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: Study which meets basic scientific principles

Objective of study:

toxicokinetics

Principles of method if other than guideline:

The absorption, distribution, metabolism and excretion (ADME) of (14)C-TEA derived radioactivity were determined in male C3H/HeJ mice following dermal application of 2000 mg/kg (neat) or, to characterize blood kinetics, intravenous (iv) injection of 1 mg/kg (14)C-TEA.

GLP compliance:

no

Radiolabelling:

yes

Species:

mouse

Strain:

C3H

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 20-30 g
- Fasting period before study: none
- Housing: individually
- Individual metabolism cages: yes
- Diet: Certified Lab Diet ad libitum
- Water: water ad libitum

Route of administration:

intravenous

Vehicle:

water

Dose / conc.:

1 other: mg/kg bw

No. of animals per sex per dose / concentration:

27 mice in total

Control animals:

not specified

Details on excretion:

Urine was the primary route of excretion of radioactivity (46-69% of total dose; 58-79% of absorbed dose) followed by feces (16-28% of total dose; 19-32% of absorbed dose). The body burden of radioactivity was 3.1% of the applied dose.

Metabolites identified:

no

Radioactivity in blood declined in a bi-exponential manner through 24-hour post-dosing with a rapid initial phase (half-life of 0.3 hr) followed by a slower terminal phase (half-life of 10 hr). Clearance of radioactivity from blood was calculated to be approximately 1.5 mL/hr/kg and the apparent volume of distribution was estimated to be approximately 11 mL.

Reference 7

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:

other: Study which meets basic scientific principles

Qualifier:

no guideline available

Principles of method if other than guideline:

The human skin penetration of triethanolamine (TEA) was measured using in vitro diffusion cell techniques.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

[(14)C]TEA

Species:

human

Type of coverage:

open

Dose:

1 %

Parameter:

percentage

Absorption:

ca. 22 %

Remarks on result:

other: 24 hrs

Remarks:

oil in water emulsion pH 8.0

Dose:

1 %

Parameter:

percentage

Absorption:

ca. 9.8 %

Remarks on result:

other: 24 hrs

Remarks:

oil in water emulsion pH 7.0

Dose:

1 %

Parameter:

percentage

Absorption:

9.6 %

Remarks on result:

other: 72 hrs

Remarks:

oil in water emulsion pH 7.0

Dose:

5 %

Parameter:

percentage

Absorption:

ca. 16.5 %

Remarks on result:

other: 24 hrs

Remarks:

oil in water emulsion pH 8.0

Dose:

5 %

Parameter:

percentage

Absorption:

ca. 5.8 %

Remarks on result:

other: 24 hrs

Remarks:

oil in water emulsion pH 7.0

Dose:

5 %

Parameter:

percentage

Absorption:

6.9 %

Remarks on result:

other: 72 hrs

Remarks:

oil in water emulsion pH 7.0

1 % dose

The mean percent of dose as radioactivity was 1.1% absorbed into the receptor fluid with an additional 20.9% penetrated into the human skin when the test material in an oil-in-water emulsion was tested at pH 8.0. The mean percent of dose as radioactivity was 0.43% absorbed into the receptor fluid with an additional 9.4% penetrated into the human skin when the test material in an oil-in-water emulsion was tested at pH 7.0. This difference with pH reflects the higher percentage of the unionized form of test material at pH 8.0.

5 % dose

The mean percent of dose as radioactivity was 1.2% absorbed into the receptor fluid with an additional 15.4% penetrated into the human skin when the test material in an oil-in-water emulsion was tested at pH 8.0. The mean percent of dose as radioactivity was 0.28% absorbed into the receptor fluid with an additional 5.5% penetrated into the human skin when the test material in an oil-in-water emulsion was tested at pH 7.0. This difference with pH reflects the higher percentage of the unionized form of test material at pH 8.0.

Reference 8

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: Study which meets basic scientific principles

Principles of method if other than guideline:

The absorption, distribution, metabolism and excretion (ADME) of (14)C-TEA derived radioactivity were determined in male C3H/HeJ mice following dermal application of 2000 mg/kg bw (neat) or, to characterise blood kinetics, intravenous (iv) injection of 1 mg/kg bw (14)C-TEA.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

mouse

Strain:

C3H

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 20-30 g
- Fasting period before study: none
- Housing: individually
- Individual metabolism cages: yes
- Diet: Certified Lab Diet ad libitum
- Water: water ad libitum

Type of coverage:

other: open or occluded

Vehicle:

other: unchanged, acetone or water

Duration of exposure:

24 hrs

Doses:

1000 mg/kg bw (acetone)
2000 mg/kg bw (no vehicle and water)

No. of animals per group:

24 (no vehicle)
3 (acetone)
3 (water)

Control animals:

no

Details on study design:

Male C3H/HeJ mice, 20-30 g, were randomly assigned to study groups and housed individually in suspended wire mesh-bottomed stainless steel cages in environmentally-controlled rooms. Certified Lab Diet and water were available ad libitum throughout the study. The blood kinetics and absorption, distribution, metabolism, excretion (ADME) of radiolabeled test material was determined following application of neat radiolabeled test material without occlusion of the application site. In addition, balance and excretion data were collected from mice dosed with test material using several other dosing scenarios: (1) test material in acetone (unoccluded)-3 mice; (2) test material neat (occluded or unoccluded); (3) test material neutralized in water (occluded)-3 mice. A site was clipped on the anterior dorsal side of all test animals 15 hours prior to dosing. Certain groups of mice were prevented from grooming this area via use of an occlusive device; a small glass ring fixed to their backs. A dosing volume of approximately 1.5 and 6 mL/kg for neat and diluted dosing solutions, respectively, was spread over the clipped skin or within the area enclosed by an attached ring (polyethylene screen glued to top of ring after dosing). Individual animals within an experiment received the same average total radioactivity (1.8-8 µCi). Those mice designated for excreta collection and sacrifice at 24 hours post-dosing were placed in Roth-style metabolism cages. Urine, feces and expired CO2 were collected at 12 and 24 hours from the 24-hour group of mice housed in metabolism cages. Following sacrifice of this group, tails were removed and livers and kidneys excised. Roth cages were washed to recover urine. Radioactivity in aliquots of excreta, cage-wash solution, liver, kidneys, skin and remaining carcass was quantified using liquid scintillation spectroscopy. Selected urine samples were subjected to ion-exchange liquid chromatography for fractionation and gas chromatography and mass spectroscopy for analysis of metabolites of test material.The absorption kinetics of dermally applied radioactivity in mice treated with radiolabeled test material neat (unoccluded) was determined. Elimination of radioactivity from blood was monitored over a 48-hour period, but not reported.

Absorption in different matrices:

Urine was the primary route of excretion of radioactivity (46-69% of total dose; 58-79% of absorbed dose) followed by feces (16-28% of total dose; 19-32% of absorbed dose). The body burden of radioactivity was 3.3%, 5.7% and 6.1% for 1000 mg/kg bw (in acetone), 2000 mg/kg bw (neat, unoccluded) and 2000 mg/kg bw (neat, occluded), respectively, of the applied dose.
The average amount of the administered dose found in the skin at the application site of mice with access to the application site ranged from 1.2 to 2.1%, while approximately 6-11% was present at this site in mice having restricted access.

Radioactivity in blood after dermal application of 2000 mg/kg bw neat TEA declined in a bi-exponential manner through 3-hour post-dosing with a rapid initial phase (half-life of 1.9 hr) followed by a slower terminal phase (half-life of 31 hr). Clearance of radioactivity from blood was calculated to be approximately 955 mL/hr/kg.

Reference 9

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: Well documented study report, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Triethanolamine uniformly radiolabeled with 14C was administered to C3H/HeJ mice intravenously or dermally to study the skin penetration of the test substance in vitro.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7H-5317

RADIOLABELLING INFORMATION
- Radiochemical purity: 97-98 % (98.6 % by liquid chromatography reanalysis)
- Specific activity: 34.7 mCi/mmol

Radiolabelling:

yes

Remarks:

14C

Species:

mouse

Strain:

other: C3H/HeJ

Sex:

not specified

Details on test animals or test system and environmental conditions:

- Source: Jackson Laboratories, Bar Harbor, ME
- Weight at study initiation: 20.4 to 38.3 g
- Fasting period before study: none
- Housing: glass Roth-style metabolism cages
- Individual metabolism cages: yes
- Diet: Purina® Certified Rodent chow #5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40-60
- Air changes (per hr): ca. 12
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

other: acetone or water

Duration of exposure:

48 hours

Doses:

1000 or 2000 mg/kg bw

No. of animals per group:

1000 mg/kg bw: 9
2000 mg/kg bw (with acetone): 48
2000 mg/kg bw (with water): 21

Control animals:

no

Details on study design:

Dermal probe - 1000 mg/kg - mice. Nine male mice received a dermal dose of 14C-TEA in acetone vehicle targeted at 1000 mg/kg. The hair on the back of each mouse was clipped immediately prior to dosing and dose applied to an area of approximately 1 square centimeter in the interscapular area using a blunted microliter syringe. Following administration of the dose, the skin at the dose site was rubbed gently with the barrel of the dosing syringe needle. The dosing volume applied dermally was 96.3 ± 3.2 % (mean ± S.D.) of the target dosing volume of 0.05 ml. The average amount of radioactivity applied was 7.95 µCi/mouse. The speciflc activity of the dosing solution was 163.5 µCi/ml. Immediately following dosing the animals were returned to their cages. The animals designated for excreta collection and sacrifice at 24 hours post-dosing (final sacrifice) were placed in glass Roth cages. Prior to dosing the animals were acclimated to Roth cages for approximately 48 hours. Blood samples were collected from all animals via orbital Sinus puncture just prior to their sacrifice by cervical dislocation in groups of three at 6, 12 and 24 hours post-dosing. Urine and feces were collected at 6 hour intervals from the mice housed in Roth cages. Following the sacrifice of this group at 24 hours, the animals were
skinned, the liver and kidneys excised, and each Roth cage washed with a solution of acetone and water. Radioactivity in excreta, cage wash solution, livers, kidneys, skin and remaining carcass were determined as described below.

Dermal administration - 2000 mg/kg - mice. Forty-eight male mice (two groups of 24 as described below) were dosed dermally with a dose of 14C-TEA targeted at 2000 mg/kg (= ca. 40-60 µl of "neat" material). These mice were dosed to determine the blood concentration-time profile for radioactivity derived from 14C-TEA. In addition, the routes and rates of excretion, the distribution
of radioactivity to liver, kidney, skin, and the remaining carcass at peak blood concentrations and at 48 hours post-dosing were determined.
The first group of twenty-four mice were dosed dermally with no effort made to restrict access to the dosed site. The dosing volume applied dermally was 104.1 ± 5.2 % (mean ± S.D.) of the target dosing volume of 60 µl. The average amount of radioactivity applied was 1.86 µCi/mouse. The hair on the back of each mouse was clipped on the day prior to dosing, and dose applied to the
skin in an area af approximately 2 square centimeters using a blunted micoliter syringe.
The second group of twenty-four mice received the dose within a glass ring adhered to the back with cyanoacrylate and covered with nylon screen, as described above. The dosing volume applied dermally was 91.3 ± 7.5 % (mean ± S.D.) of the target dosing volume of 45 µl. The average amount of radioactivity applied was 2.76 µCi/mouse.

Immediately following dosing the animals from both groups scheduled for sacrifice at 48 hours post-dosing were placed in Roth cages for the separation of urine and feces. Urine and feces were collected at 6 and 12 hours postdosing (if available) and every 12 hours thereafter. Blood samples were collected from all animals via orbital Sinus puncture just prior to their sacrifice by cervical dislocation in groups of three at 0.083, 0.167, 0.5, 1, 2, 4, 6, 12, 24 and 48 hours post-dosing.

Following the sacrifice of the group killed at 48 hours, the skin from the dose site was excised, the remaining skin removed, the liver and kidneys excised, and each Roth cage was washed with a solution of acetone and water. The glass rings used to restrict access to the dosed site were removed from the skin and washed with water to recover any residual radioactivity on the rings. Radioactivity in excreta, cage wash solution, ring wash solution, liver, kidneys, skin and remaining carcass were determined as described below. The
concentration of radioactivity in the liver, kidney, skin from the dosed site, skin remote from the dosed site, and the remaining carcass was also determined for the animals sacrificed at 3 hours post-dosing which had access to the dosed site. Three hours post-dosing was the time where the blood concentTation of 14C-TEA derived radioactivity peaked.

Dermal probe - 2000 mg/kg - mice. Prior to the studies described above a dermal probe was carried out where six male mice were dosed dermally with a dose of 14C-TEA targeted at 2000 mg/kg (about 60 µl "neat" material). The purpose of probe was to examine if there were any demonstrable differences between results when access to the dermal dose site was restricted. The dosing
volume applied dermally was 104.2 ± 6.5 % (mean ± S.D.) of the target dosing volume of 60 µl. The average amount of radioactivity applied was 2.56 µCi/mouse. The hair on the back of each mouse was clipped on the day prior to dosing, and dose applied to the skin in an area of approximately 1.75 square centi.meters using a blunted microliter syringe. Three mice had the dose applied within a small glass ring (1.5 cm in diameter, 0.5 an in height) which had been adhered to the skin using cyanoacrylate about 15 hours prior
to dosing. After dosing, the ring was covered with a rigid piece of nylon screen attached with cyanoacrylate to restrict the animals access to the dosed skin. The remaming three mice did not have glass rings adhered to skin, but had free access to the dosing site. Immediately following dosing, the animals were placed in Roth cages for the separation and collection of urine and feces. Urine was collected a 6,12 and 24 hours post-dosing and feces was collected at 12 and 24 hours post-dosing. Blood samples were taken via the retro-orbital sinus just prior to sacrifice by cervical dislocation at 24 hours post-dosing. Following sacrifice, the skin from the dosed site was excised, the remaining skin removed, the liver and kidneys excised, and each Roth cage was washed with a solution of acetone and water. The glass rings used to restrict access to the dosed site were removed from the skin and washed with water to recover any residual radioactivity on the rings. Radioactivity in excreta, cage wash solution, ring wash solution, livers, kidneys, skin and remaining carcass were determined as described below.

Dermal administration using water as a vehicle -2000 mg/kg - mice. In order to investigate the effect of water as a dosing vehicle for TEA, twenty-one male mice were dosed dermally with a dose of 14C-TEA in aqueous solution with the pH of the dosing solution adjusted to neutrality. The applied dose was targeted at 2000 mg/kg with a target dosing volume of 200 µl. Prior to dosing
the hair on the back of the animals was dlipped. The dose was applied to the skin within a glass ring (as described above) which was adhered to the back with cyanoacrylate and covered with polyethylene screen to restrict access to the dose site and to aid in containing this larger volume of dose at one site. The rings were attached about 15 hours prior to dosing with the exception of
three animals where the rings were attached on the day of dosing. The mice were anesthetized with methoxyflurane during the attachment of the rings. The dosing volume applied dermally was 97.6 ± 7.6 % (mean ± S.D.) of the target dosing volume. The average amount of radioactivity applied was 8.2 µCi / mouse. Blood samples were taken from each animal via orbital Sinus puncture just prior to their sacrifice in groups of three at 1, 2, 4, 6, 12, 24 and 48 hours postdosing. Animals scheduled to be sacrificed at 48 hours were placed in Roth cages immediately after dosing. Following the sacrifice of the 48 hour group, the skin from the dose site was excised, the remaining skin removed, the liver and kidneys excised, and each Roth cage was washed with a solution of acetone and water. The glass rings used to restrict access to the dosed site were removed from the skin and washed with water to recover any residual radioactivity on the rings. Radioactivity in excreta, cage wash solution, ring wash solution, liver, kidneys, skin and remaining carcass were determined as described below.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

Upon dermal application, triethanolamine was rapidly absorbed by the mouse skin. The dermal absorption is 94.5% after application of 1000 mg/kg bw and 97.8% after application of 2000 mg/kg bw.

Radioactivity was eliminated from mouse blood with a half-life time of 10 -18 hours following a first-order-process. 65 % of the applied dose was recovered from urine, 25 % from faeces.

Upon dermal application, triethanolamine was rapidly absorbed by the mouse skin while dermal absorption by rats was much lower. Radioactivity was was eliminated from mouse blood with a half-life time of 10 -18 hours following a first-order-process. 65 % of the applied dose was recovered from urine, 25 % from faeces.

Pharmacokinetics and metabolism of triethanolamine was found to be dose-independent in mice. 24 hours after dosing the remaining activity was found to be 3.6 % in mice after intravenous injection, 4.5 % after dermal application of 1000 mg/kg bw and 7.8 % after dermal application of 2000 mg/kg. The remaining activity was mainly found in liver and kidney.

Reference 10

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Triethanolamine uniformly radiolabeled with 14C was administered to C3H/HeJ mice intravenously or dermally to study the skin penetration of the test substance in vitro.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7H-5317

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 97-98 % (98.6 % by liquid chromatography reanalysis)
- Specific activity: 34.7 mCi/mmol

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Weight at study initiation: 231 to 247 g
- Fasting period before study: none
- Housing: glass Roth-style metabolism cages
- Individual metabolism cages: yes
- Diet: Purina® Certified Rodent chow #5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40-60
- Air changes (per hr): ca. 12
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Duration of exposure:

48 hours

Doses:

1000 mg/kg bw

No. of animals per group:

3

Control animals:

no

Details on study design:

About 48 hours prior to dosing, indwelling cannulas were surgically implanted into the jugular vein of the rats while they were under methoxyflurane anesthesia using a modification of the method described by Harms and Ojeda, 1974. Approximately, 16 hours prior to dosing, die animals were anesthetized with methoxyflurane and the skin on the back of the rats was shaved using Ivory® soap and a straight razor. The skin was dried and a glass ring (as previously described) was attached to the skin using cyanoacrylate. At the time of dosing, die animals were anesthetized with methoxyflurane and a dosing volume targeted at 200 µl of "neat" 14C-TEA was applied within the ring. The ring was then covered with a round glass cover slip attached with cyanoacrylate to restrict the animals access to the dose site while grooming and to aid in containing this large volume of dose at one site. The dosing volume applied dermally was 96.8 ± 0.3 % (mean ± S.D.) of the target dosing volume. The average amount of radioactivity applied was 4.42 µCi/rat.
Immediately following dosing the animals were placed in glass Roth cages for the separation of urine and feces. Urine and feces were collected at 6, 12, 24 and 48 hours post-dosing. Blood samples (= 0.2 ml) were taken via the indwelling jugular cannulas at 0.5, 1, 2, 4, 6, 12, 24 and 36 hours post-dosing. An additional blood sample was drawn from one animal at 31 hours post-dosing. At 48 hours post-dosing the animals were anesthetized with methoxyflurane and sacrificed by exsanguination via cardiac puncture. Following sacrifice, the skin from the dose site was excised, the remaining skin removed, the liver and kidneys excised, and each Roth cage was washed with a solution of acetone and water. The glass rings used to restrict access to the dosed site were removed from the skin and washed with water to recover any residual radioactivity on thie rings. Radioactivity in excreta, cage wash solution, ring wash solution, liver, kidneys, skin and remaining carcass were determined.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

Upon dermal application, the dermal absorption of triethanolamine was less extensive and much slower than in mice. Absorption values have not been provided.

Total recovery:

The urinary interval excretion data show that the percentage of the administered dose found in the urine per collection interval (0-6, 6-12, 12-24 and 24-48 hrs) was 3% or less.
The level of radioactivity was non-detectable (≤2x background) in the blood samples taken at 0.5, 1, 2, 4 and 6 hrs post-dosing

Conversion factor human vs. animal skin:

No data

As long as the glass ring and coverslip attached to the skin to restrict the animals access to the dosed was intact, the results are as indicated above. Once the integrity of the glass ring or coverslip failed, the dose was leaked to other areas of the skin and ingestion of the dose incidental to grooming could occur. Indeed, the percentage radioactivity found in the urine increased markedly in each as soon as the device for restricting access to the dosed site failed. In the blood, radioactivity was only detectable at sampling times which were after the integrity of the glass ring restricting access to the dosed site had failed. This suggests that most of the blood radioactivity found may have been a result of ingestion incidental to grooming after access to the test material on the skin was obtained. Data were not sufficient to construct a meaningful blood concentration-time profile.

Additional information

1. Physical-chemical properties

TEA (MW 149.2 g/mol) is a liquid with a measured melting point of 20.5°C, a measured boiling point of 336.1°C at 1013.25 hPa, a measured vapour pressure of 0.00029 hPa at 21°C, and a dissociation constant (pKa) of 7.86 at 25°C. The octanol-water partition coefficient (log Pow) is -2.3 at 25°C, and the substance is fully miscible with water.

2. Data from acute and repeated dose toxicity studies

Acute toxicity data indicate low toxicity: in rats the oral LD50 was 6400 mg/kg bw, no mortality was observed at or below 5000 mg/kg bw. Clinical signs (elevated respiration, anancasm to chew, apathy, reduced grooming) disappeared 2 days after dosing, and gross pathology at necropsy revealed no abnormalities (BASF AG, 1966). In an acute dermal toxicity study in rabbits, no mortality was observed up to the limit concentration and the LD50 was established to be > 2000 mg/kg bw (TSCATS, 1989). Due to its extremely low vapour pressure, exposure to TEA vapour is very unlikely. One report stated that whole-body exposure of rats to an atmosphere saturated with TEA vapour (concentration not given) at 20°C for 8 hours failed to cause any deaths, therefore no LC50 value was established (BASF AG, 1966).

In an oral repeated dose study, rats were administered 0 - 1000 mg/kg bw/day in the diet for 91 days. Since no adverse effects were observed, the NOAEL was established to be 1000 mg/kg bw/day (TSCATS, 1989). In a sub-chronic dermal toxicity study, rats were treated with 0 - 2000 mg/kg bw/day on the skin for 90 days (Battelle Columbus Laboratories, 1987a). At the highest doses, decreases in body weight, irritation and inflammation at the site of application were observed - ranging from minimal acanthosis at the lower doses to chronic active inflammation, erosion and ulceration in higher dose groups - accompanied by haematologic changes. NOAELs for local effects were determined to be 125 and 250 mg/kg bw/day for males and females, respectively. The NOAEL for systemic effects was established at 125 mg/kg bw/day, based on renal effects (i.e. increased kidney weight). Similar effects were observed in a sub-chronic dermal toxicity study in mice, receiving 0 - 4000 mg/kg bw/day TEA on the skin for 90 days (Battelle Columbus Laboratories, 1987b). The kidneys were identified as the target organ at lower doses, accompanied by increased liver weights at the top dose level. Dermal irritation and inflammation was noted at the site of application. In an 28 -day inhalation toxicity study in rats, exposed to 0 - 0.5 mg/L TEA for 6 hours/day and 5 hours/week, the NOAEC for systemic effects was established at 0.5 mg/L since no adverse systemic effects were observed. The NOAEC for local effects (laryngeal inflammation) was determined to be 0.02 mg/L for females; since slight inflammation was still observed in males, this concentration was designated the LOAEC for local effects in males (BASF AG, 1993).

3. Absorption, distribution, metabolism, excretion

Studies in experimental animals indicated that TEA is absorbed through the skin. No data on oral and inhalation exposure is available. Besides data regarding the dermal route, data on the i.v. route is also available. Differences in the rate of absorption between rats and mice have been described regarding dermal exposure. In mice, most of the topically applied¹⁴C-TEA is absorbed, and only 2% to 11% is detected at the site of application after 48 hours (Dow 1988,1989; Stott, 2000). The dermal absorption of TEA in rats was less extensive and much slower than in mice (Dow, 1988,1989). An absorption, distribution, metabolism, and excretion study by the NTP (2004) found that after 72 hours of exposure, only 20% to 30% of the applied dermal dose of TEA (68 or 276 mg/kg) was absorbed in rats and 60% to 80% was absorbed in mice (79 or 1120 mg/kg). These differences in absorption have been attributed either to the different doses used in comparative studies or to species-specific factors. No differences in tissue distribution were noted after i.v. or dermal exposure (NTP, 2004).

The elimination of¹⁴C-TEA-derived radioactivity from the blood of mice after a 1 mg/kg intravenous injection displays two-phase elimination kinetics with an initial rapid distribution phase (0.3-0.6 hour half-life) followed by a slower elimination phase (10-hour half-life) (Dow, 1988,1989; Stott, 2000). Radioactivity in blood after dermal application of 2000 mg/kg neat TEA declined in a bi-exponential manner through 3-hour post-dosing with a rapid initial phase (half-life of 1.9 hr) followed by a slower terminal phase (half-life of 31 hr)(Stott, 2000). Both rats and mice rapidly excreted the absorbed dose, primarily in urine (followed by faeces) after i.v. and dermal exposure. Regarding dermal exposure, in rats, less than 1% of the dose was present in the tissue samples (except the dose site) 72 hours after treatment; the heart, kidney, liver, lung, and spleen contained elevated concentrations of radiolabel relative to blood (NTP, 2004).

 

In addition to animal studies, human skin penetration of TEA was tested in vitro using diffusion cell techniques (Kraeling, 2003). Oil-in-water emulsions containing 1% or 5%¹⁴C-TEA were added to the stratum corneum side of 200-300 µm thick human skin sections and penetration of radioactivity into and through the skin (into a receptor fluid, sampled up to 24 hours after application) was determined. At pH 8.0, 1.1 and 1.2% of the dose was absorbed into the receptor fluid with a total penetration of 22.0 and 16.5% for 1 and 5% TEA, respectively. At pH 7.0, 0.43 and 0.28% was absorbed into the receptor fluid with a total penetration of 9.8 and 5.8% after 24 hours for 1 and 5% TEA, respectively. After 48 hours at pH 7.0, 0.68 and 0.60% was absorbed into the receptor fluid with a total penetration of 9.6 and 6.9%, for 1 and 5% TEA respectively. This pH-related difference reflects the higher percentage of unionised test material at pH 8.0.