L-Aspartic acid, N,N'-1,2-ethanediylbis-, sodium salt (1:3)

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 to 7 May 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

only one low dose used

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl: (WI)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada, St-Constant, Quebec
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 191-303 g; females 173-253 g
- Fasting period before study: no data
- Housing: glass metabolism cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4 May 1993 To: 7 May 1993

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: 7.6 cm2
- % coverage: no data
- Type of wrap if used: glass chamber applied with Permabond 102 adhesive
- Time intervals for shavings or clipplings: before dosing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test site washed at study termination at 72 h, and included with the rinsings from the dermal chamber for counting of radioactivity
- Time after start of exposure: 72 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.2 mL aqueous solution
- concentration (if solution): males, 4.14 mg/kg bw (66.7 uCi/kg bw); females, 5.12 mg/kg bw (84.0 uCi/kg bw )

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied: 0.2 mL

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Duration and frequency of treatment / exposure:

72 h

Dose / conc.:

4.14 mg/kg bw/day (nominal)

Remarks:

Males, 4.14 mg/kg bw (66.7 uCi/kg bw)

Dose / conc.:

5.12 mg/kg bw/day (nominal)

Remarks:

females, 5.12 mg/kg bw (84.0 uCi/kg bw )

No. of animals per sex per dose / concentration:

5/sex test group
1 male acted as a control

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: selected to provide sufficient material to determine the distribution, elimination and mass balance of radioactivity
- Rationale for animal assignment (if not random): random

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, expired air, adipose tissue, brain, bone marrow, femur bone, gonads, heart, GI (combined stomach, large and small intestine), GI contents, kidneys, liver, lungs, muscle (right leg adductor), pancreas, spleen, carcass, skin from test site and three adjacent sites.

- Time and frequency of sampling: urine, faeces and cage washes were collected at 24, 48 and 72 h; expired air was trapped in potassium hydroxide and collected at 12, 24, 36, 48 and 72 h. Tissues and organs were sampled for radioactivity at study termination (72 h).

Statistics:

Bartlett's test and t-tests were used to determine any gender differences in the recovery of radioactivity. A in-transformation on the percent of dose was used to correct the non homogeneity of the variances when appropriate. For the skin test site, adjacent skin and dermal cell rinse a one-way ANOVA was used to compare the gender effect. The parameters tested were: radioactivity recovered in the tissues, urine, faeces, expired air, skin dose site, adjacent skin, dermal cell rinse and total recovered radioactivity.

Preliminary studies:

none

Type:

absorption

Results:

After 72 h, 11.1% of applied dose was absorbed in males; 5% absorbed in females

Type:

distribution

Results:

the combined mean blood and tissue (including carcass) recovery was 2.34% in males and 1.45% in females

Type:

excretion

Results:

about 9% of the applied dose was excreted in males and about 4% in females during 72 h exposure

Details on absorption:

Only about 11% of the applied dose was absorbed in males and 5% in females during the 72 h exposure period.

The total recovery of radioactivity was 59.1% in males and 62.8% in females. This mainly represented unabsorbed material found at the skin treatment site (about 40% of applied dose) and dermal chamber washings (about 10%).

Details on distribution in tissues:

Low amounts of radioactivity were detected in the organs and tissues examined, ranging from 0% to up to 1.2% in the male carcass. See table 1 for details.

Details on excretion:

Less than 9% of applied dose recovered in the excreta (urine, faeces, cage wash and expired air). See tables 1 and 2 for details.

Metabolites identified:

not measured

Table 1. Total recovery (mass balance) of radioactivity after dermal exposure of rats

Sample	Males (% of dose)	Females (% of dose)
Skin test site	38.5 ± 9.38	43.9 ± 11.4
Skin adjacent site	0.908 ± 0.829	0.916 ± 1.117
Dermal cell rinse	8.60 ± 3.36	12.8 ± 7.42
Urine	2.26 ± 1.63	0.521 ± 0.578
Faeces	4.90 ± 3.79	2.06 ± 2.69
Expired air	0.643 ± 0.413	0.340 ± 0.186
Cage wash	0.954 ± 0.879	0.805 ± 0.893
Blood	0.003 ± 0.006	0.000 ± 0.000
Liver	0.025 ± 0.021	0.023 ± 0.022
Kidneys	0.012 ± 0.012	0.004 ± 0.003
Heart	0.000 ± 0.000	0.001 ± 0.002
Lungs	0.000 ± 0.000	0.007 ± 0.010
Pancreas	0.002 ± 0.002	0.002 ± 0.001
Spleen	0.000 ± 0.000	0.004 ± 0.005
Brain	0.000 ± 0.000	0.000 ± 0.000
Testes/ovaries	0.000 ± 0.001	0.001 ± 0.002
Muscle	0.014 ± 0.031	0.000 ± 0.000
Epididymal fat	0.014 ± 0.018	0.121 ± 0.156
Bone marrow	0.000 ± 0.000	0.000 ± 0.000
Bone femur	0.000 ± 0.000	0.001 ± 0.002
GI tract	0.173 ± 0.297	0.184 ± 0.233
GI contents	0.896 ± 1.38	0.314 ± 0.261
Carcass	1.20 ± 0.914	0.794 ± 0.738
Total	59.1 ± 8.03	62.8 ± 18.6

Table 2. Mean cumulative recovery of radioactivity in excreta after dermal exposure

Time (h)	Males (% of dose)					Females (% of dose)
	Urine	Faeces	Expired air	Cage wash	Total	Urine	Faeces	Expired air	Cage wash	Total
0-12	a	a	0.086 ± 0.014	a	0.086 ± 0.014	a	a	0.087 ± 0.035	a	0.087 ± 0.035
0-24	1.51 ± 1.59	0.884 ± 1.18	0.145 ± 0.05	0.492 ± 0.627	3.03 ± 3.36	0.355 ± 0.26	0.168 ± 0.212	0.111 ± 0.043	0.032 ± 0.049	0.577 ± 0.438
0-36	1.51 ± 1.59	0.884 ± 1.18	0.251 ± 0.116	0.492 ± 0.627	3.14 ± 3.34	0.355 ± 0.26	0.168 ± 0.212	0.162 ± 0.079	0.032 ± 0.049	0.628 ± 0.470
0.45	1.74 ± 1.59	2.44 ± 1.79	0.437 ± 0.244	0.730 ± 0.722	5.35 ± 3.15	0.349 ± 0.322	0.998 ± 1.63	0.226 ± 0.152	0.627 ± 0.692	2.20 ± 2.66
0-72	2.26 ± 1.63	4.90 ± 3.79	0.643 ± 0.413	0.954 ± 0.879	8.76 ± 4.87	0.521 ± 0.578	2.06 ± 2.69	0.340 ± 0.186	0.805 ± 0.893	3.72 ± 4.28

a, no sample collected

Conclusions:

In a GLP study, similar to OECD Guideline 417, only about 11% of the applied dermal dose of radiolabelled-trisodium EDDS was considered absorbed in males and 5% in females following a 72-h exposure period. Less than 9% was recovered in the excreta (urine, faeces, cage wash and expired air). Low amounts of radioactivity were detected in the organs and tissues examined, ranging from 0% to up to 1.2% in the male carcass.

Executive summary:

In a GLP study conducted according to a protocol similar to OECD Guideline 417, the absorption, distribution and elimination of 14C-trisodium EDDS was determined in male and female Wistar rats.

The radiolabelled test substance was applied (in water) at 4-5 mg/kg bw in a glass chamber to the shaved skin of five rats of each sex for 72 h. Urine, faeces, cage washings and expired air were collected at intervals throughout the study period and the radioactive content was measured. At study termination, levels of radioactivity were determined in adipose tissue, brain, bone marrow, femur bone, gonads, heart, gastrointestinal tract, gastrointestinal contents, kidneys, liver, lungs, muscle, pancreas, spleen, carcass, skin from the treatment site and adjacent sites.

The total recovery of radioactivity from all sources was 59.1% in males and 62.8% in females. This mainly represented unabsorbed material found at the skin treatment site (about 40%) and in the dermal chamber washings (about 10%). Only about 11% of the applied dose was considered absorbed in males and 5% in females during the 72-h exposure period, with less than 9% recovered in the excreta (urine, faeces, cage wash and expired air). Low amounts of radioactivity were detected in the organs and tissues examined, ranging from 0% to up to 1.2% in the male carcass.

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 to 29 April 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

only one low dose used

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Crl: (WI)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada, St-Constant, Quebec
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 191-303 g; females 173-253 g
- Fasting period before study: no data
- Housing: in glass metabolism cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 26 April 1993 To: 29 April 1993

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solution pre-prepared at the appropriate concentration by the sponsor

HOMOGENEITY AND STABILITY OF TEST MATERIAL: no data

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

Gavaged with 1 mL to give about 10 uCi/rat and 0.5 mg/rat (about 3 mg/kg bw) of 14C-[S,S]-EDDS trisodium salt

No. of animals per sex per dose / concentration:

5/sex

Control animals:

no

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: selected to provide sufficient material to determine the distribution, elimination and mass balance of radioactivity
- Rationale for animal assignment (if not random): random

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, expired air, adipose tissue, brain, bone marrow (femur), femur bone, gonads, heart, GI (combined stomach, large and small intestine), GI contents, kidneys, liver, lungs, muscle (right leg adductor), pancreas, spleen, carcass
- Time and frequency of sampling: urine, faeces and cage washes were collected at 24, 48 and 72 h; expired air was trapped in potassium hydroxide and collected at 12, 24, 36, 48 and 72 h. Tissues and organs were sampled for radioactivity at study termination at 72 h.

Statistics:

Bartlett's test and t-tests were used to determine any gender differences in the recovery of radioactivity. A ln-transformation on the percent of dose was used to correct the non-homogeneity of the variances when appropriate.

Preliminary studies:

none

Type:

absorption

Results:

after 72 h, at least 5% of the dose was considered absorbed

Type:

distribution

Results:

after 72 h, a total of about 0.15% of the dose was recovered in the blood and organs (including carcass)

Type:

excretion

Results:

after 72 h, about 75% of the dose was excreted in the faeces, and about 2 and 3% in the urine and expired air, respectively

Details on absorption:

Only about 5% of the dose was absorbed from the gastrointestinal tract as shown by the radioactivity recovered from urine, expired air and recovered in the tissues during the 72 h exposure period. The cage wash may have contained some further absorbed material (excreted as urine). See Tables 1 and 2 for further details.

Details on distribution in tissues:

The total radioactivity recovered from the organs and tissues examined are given in Table 1. The combined mean radioactivity content of blood and tissues (including carcass) was 0.136 and 0.153% in males and females, respectively.

Details on excretion:

Radioactivity was excreted mainly in the faeces and occurred rapidly with about 62% and 71% of the dose being recovered in faeces in males and females, respectively, during the first 24 h. In total, during the 72-h collection period, faecal excretion accounted for about 75% of the administered dose in males and females. Only a small fraction of the dose was recovered in the urine (about 1.7%) and expired air (3.25%). See Tables 1 and 2 for further details.

Metabolites identified:

not measured

Table 1. Total recovery (mass balance) of radioactivity in excreta and tissues

Sample	Males (% of dose)	Females (% of dose)
Urine	1.87 ± 1.58	1.49 ± 0.200
Faeces	75.6 ± 6.91	75.2 ± 7.92
Expired air	3.35 ± 2.21	3.19 ± 0.803
Cage wash	3.37 ± 2.86	9.38 ± 7.55
Blood	0.0041 ± 0.0093	0.000 ± 0.000
Liver	0.0424 ± 0.0353	0.037 ± 0.014
Kidneys	0.0092 ± 0.0084	0.004 ± 0.001
Heart	0.0003 ± 0.0008	0.000 ± 0.001
Lungs	0.0007 ± 0.0015	0.000 ± 0.000
Pancreas	0.0007 ± 0.0016	0.001 ± 0.002
Spleen	0.005 ± 0.0011	0.001 ± 0.002
Brain	0.0015 ± 0.0034	0.000 ± 0.000
Testes/ovaries	0.0010 ± 0.0023	0.000 ± 0.000
Muscle	0.0000 ± 0.0000	0.000 ± 0.000
Adipose tissue	0.0283 ± 0.0127	0.017 ± 0.026
Bone marrow	0.0000 ± 0.0000	0.000 ± 0.000
Bone femur	0.0000 ± 0.0000	0.000 ± 0.000
GI tract	0.0358 ± 0.0478	0.013 ± 0.012
GI contents	0.0345 ± 0.0771	0.000 ± 0.000
Carcass	0.114 ± 0.141	0.080 ± 0.083
Total	84.4 ± 1.52	89.5 ± 2.76

Table 2. Percentage recovery of radioactivity in excreta

	% of dose					Cumulative % of dose
Period (h)	Urine	Faeces	Cage wash	Expired air	Total	Urine	Faeces	Cage wash	Expired air	Total
Males
0-12	a	a	a	1.2228 ± 0.2709	1.2228 ± 0.2709	a	a	a	1.2228 ± 0.2709	1.2228 ± 0.2709
12-24	1.3003 ± 0.4784	62.2836 ± 26.9438	2.2841 ± 2.1168	0.7793 ± 0.3835	66.6474 ± 26.2117	1.3003 ± 0.4784	62.2836 ± 26.9438	2.2841 ± 2.1168	2.0021 ± 0.1764	67.8702 ± 26.3440
24-36	a	a	a	0.6048 ± 0.8138	0.6048 ± 0.8138	1.3003 ± 0.4784	62.2836 ± 26.9438	2.2841 ± 2.1168	2.6069 ± 0.9824	68.4750 ± 25.5306
36-48	0.4169 ± 0.8032	8.7869 ± 11.8536	0.4881 ± 0.6665	0.4913 ± 0.7762	10.2732 ± 13.9894	1.7173 ± 1.2524	71.1606 ± 15.3503	2.7722 ± 2.4431	3.0982 ± 1.7566	78.7482 ± 12.0645
48-72	0.1532 ± 0.3335	4.3958 ± 8.6421	0.5985 ± 1.3383	0.2484 ± 0.4581	5.3959 ± 10.7689	1.8705 ± 1.5803	75.5564 ± 6.9090	3.3707 ± 2.8555	3.3466 ± 2.2133	84.1441 ± 1.7590
Females
0-12	a	a	a	1.1357 ± 0.1808	1.1357 ± 0.1808	a	a	a	1.1357 ± 0.1808	1.1357 ± 0.1808
12-24	1.2509 ± 0.1869	70.6335 ± 5.9226	9.0715 ± 7.3306	1.4762 ± 0.4461	82.4320 ± 3.5779	1.2509 ± 0.1869	70.6335 ± 5.9226	9.0715 ± 7.3306	2.6119 ± 0.5366	83.5677 ± 3.6176
24-36	a	a	a	0.3144 ± 0.1596	0.3144 ± 0.1596	1.2509 ± 0.1869	70.6335 ± 5.9226	9.0715 ± 7.3306	2.9263 ± 0.6461	83.8821 ± 3.5258
36-48	0.2609 ± 0.2946	3.9687 ± 3.8809	0.2423 ± 0.3216	0.2029 ± 0.1229	4.6226 ± 3.8030	1.4596 ± 0.1392	74.6022 ± 7.7342	9.3138 ± 7.5307	3.1292 ± 0.7494	88.5047 ± 2.7321
48-72	0.0289 ± 0.0647	0.6468 ± 0.2785	0.0622 ± 0.0956	0.0614 ± 0.0666	0.7993 ± 0.3595	1.4885 ± 0.2003	75.2490 ± 7.9168	9.3760 ± 7.5495	3.1906 ± 0.8025	89.3041 ± 2.7273

a: not sampled

Conclusions:

In a GLP study, similar to OECD Guideline 417, a single dose of radiolabelled trisodium EDDS (about 2-3 mg/kg bw/day) administered by gavage to male and female rats was rapidly eliminated, mainly in the faeces (about 75% of the administered dose), with at least 5% considered absorbed from the gastrointestinal tract.

Executive summary:

In a GLP study conducted according to a protocol similar to OECD Guideline 417, the absorption, distribution and elimination of 14C-labelled trisodium EDDS was determined in male and female Wistar rats.

The radiolabelled test substance was administered by single oral gavage (in water) to five rats of each sex at a dose level of 0.5 mg/rat (about 2-3 mg/kg bw) and the animals placed in individual metabolism cages. Urine, faeces and cage washes were collected at 24, 48 and 72 h and expired air was collected in 12, 24, 36, 48 and 72 h samples. At study termination (72 h), blood samples were taken and plasma separated and selected tissues and gastrointestinal contents were collected and the levels of radioactivity were determined by liquid scintillation counting (after solubilising the tissues as necessary).

Trisodium EDDS was rapidly excreted, mainly in the faeces. In the first 24 h, approximately 62 and 71% of the administered dose, respectively in males and females, was excreted in the faeces, and after 72 h about 75% was excreted in the faeces. Overall, at least 5% of the test substance was absorbed from the GI tract as determined by the amount of radioactivity in urine, expired air and in the tissues. Some further absorbed material may have been found in the cage wash and possibly any faeces produced after 36 hr. The combined mean radioactivity content of blood and tissues (including carcass) was 0.136 and 0.153% of the administered dose in males and females, respectively. No statistically significant gender differences were evident in the absorption, distribution or excretion patterns.

In conclusion, at least 5% of an administered dose of trisodium EDDS was considered absorbed from the gastrointestinal tract, with most being excreted in the faeces within the first 24 h after administration.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 September to 01 October 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

Principles of method if other than guideline:

In a study to determine if the test substance reached the bone marrow under similar conditions to those used in in vivo cytogenetic assays, radiolabelled trisodium EDDS was given by oral gavage to female rats and three animals necropsied at various time points over a 72-h period. Radioactivity was measured in the blood, plasma, liver, kidney, ovaries and bone marrow

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 190-250 g
- Fasting period before study: no
- Housing: hanging stainless steel metabolism cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: From: 28 September 1993 To: 1 October 1993

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 16.2 g of unlabelled trisodium EDDS were dissolved in distilled water, then 31 g of the radiolabelled test substance added, mixed and distilled water added to a final weight of 150 g of the dose solution.


HOMOGENEITY AND STABILITY OF TEST MATERIAL: fully soluble in water

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

2 053 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

27 females all received the same dose, three animals each were killed at 2, 8, 15, 24, 32, 37, 48, 56 and 72 h after dosing

Control animals:

no

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: dose selected based on the high dose proposed in an in vivo cytogenetic assay to determine the distribution of the test substance in the bone marrow

Details on dosing and sampling:

PHARMACOKINETIC STUDY (distribution)
- Tissues and body fluids sampled : blood, plasma, ovaries, kidneys, liver and bone marrow
- Time and frequency of sampling: 2, 8, 15, 24, 32, 37, 48, 56 and 72 h post exposure

Statistics:

none

Type:

distribution

Results:

Mean peak bone marrow radioactivity level was about 14 ug/g at the 24-h time point, declining slowly thereafter to about 5 ug/g by 72 h

Details on absorption:

no data

Details on distribution in tissues:

Highest levels of radioactivity were found in the kidney and liver (peaking within 8 h post-dose). No distinct rise to peak radioactivity was seen in any of the tissues analysed. Plasma radioactivity was highest at 8 h post-dose, and blood and ovary radioactivity at 15 h post-treatment, and then slowly decreased. Liver, kidney and bone marrow levels were highest at 2, 8 and 24 h, respectively, then were slowly reduced (see Table 1).

Details on excretion:

not examined

Metabolites identified:

not measured

Table 1. Mean levels of radioactivity in tissues

	Mean ug trisodium EDDS/g tissue
	2 h	8 h	15 h	24 h	32 h	37 h	48 h	56 h	72 h
Blood	8.0	5.5	13.0	6.7	8.7	3.8	3.9	4.4	2.7
Plasma	5.0	9.4	9.3	5.6	5.8	3.9	4.0	2.5	2.1
Ovaries	6.1	5.3	6.7	5.9	6.1	3.7	6.0	4.1	3.2
Kidneys	18.0	26.0	20.0	13.0	21.0	16.0	14.0	10.0	9.0
Liver	17.0	16.0	15.0	15.0	14.0	8.8	13.0	9.1	6.0
Bone marrow	9.2	4.9	8.0	14.0	13.0	6.7	10.0	7.2	5.1

Other findings: diarrhoea was observed in all animals at the 15-h necropsies, but was absent at 24 h onwards. Mean body weight had decreased by about 4% of the pre-dose weight by the 48-h necropsy but had almost returned to pre-dose weight at 72 h.

Conclusions:

In a study to determine whether orally administered trisodium EDDS reached the bone marrow, a single gavage dose (2053 mg/kg bw) of the radiolabelled test substance to female rats was detected in all the tissues analysed (blood, plasma, ovaries, kidneys, liver and bone marrow) at low levels throughout the 72 h time course, peaking within 24 h post-dose and decreasing slowly thereafter.

Executive summary:

In a study to determine whether trisodium EDDS would reach the bone marrow under similar conditions to those used in an in vivo cytogenetics assay, a single dose of the radiolabelled test substance was given by gavage to groups of 3 female rats per time point at 2053 mg/kg (in water) and the animals necropsied at 2, 8, 15, 24, 32, 37, 48, 56 and 72 h post exposure. Radioactivity was measured in the blood, plasma, liver, kidneys, ovaries and bone marrow (femur).

Highest levels of radioactivity were found in the kidney and liver (peaking within 8 h post-dose). No distinct rise to peak radioactivity was seen in any of the tissues analysed. Plasma radioactivity was highest at 8 h post-dose, and blood and ovary radioactivity at 15 h post-treatment, and then slowly decreased. Liver, kidney and bone marrow levels were highest at 2, 8 and 24 h, respectively, then were slowly reduced.

In conclusion, radiolabelled trisodium EDDS dosed orally to rats at 2053 mg/kg was detected at low levels in all the tissues analysed (blood, plasma, ovaries, kidneys, liver and bone marrow) throughout the 72-h time course, peaking within 24 h post-dose and decreasing slowly thereafter. Results of this study demonstrate that bone marrow is exposed to EDDS and/or its metabolites following oral (gavage) dosing under conditions similar to those employed in in vivo cytogenetic studies.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 to 27 August 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

Principles of method if other than guideline:

In a study to determine if the test substance reached the bone marrow under similar conditions to those used in in vivo cytogenetic assays, radiolabelled trisodium EDDS was given by oral gavage to male rats and three animals necropsied at various time points over a 72-h period. Radioactivity was measured in the blood, plasma, liver, kidney, testes and bone marrow.

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 300 +/- 25 g
- Fasting period before study: no
- Housing: stainless steel metabolism cages with wire mesh bottoms
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: 23 August 1993 To: 27 August 1993

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 21.5 g of unlabelled trisodium EDDS added to 110 g distilled water and dissolved. Then 40.3 mg of the radiolabelled test substance added and made up to 200 g with distilled water. Final activity = 3.2 uCi/g


HOMOGENEITY AND STABILITY OF TEST MATERIAL: no data. Expiry date 3 days after preparation

Duration and frequency of treatment / exposure:

single dose

Dose / conc.:

2 106 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

27 males all received the same dose, three animals each were killed at 2, 4, 8, 15, 24, 32, 48, 56 and 72 h after dosing

Control animals:

no

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: the objective of this study was to determine the distribution of the test substance to bone marrow
- Rationale for animal assignment (if not random): no data

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, plasma, testes, kidneys, liver, bone marrow (femur). Urine and faeces were collected and frozen for possible future analysis (no results on excretion are given in the report)
- Time and frequency of sampling: three animals each killed at 2, 4, 8, 15, 24, 32, 48, 56 and 72 h after dosing

Statistics:

no data

Type:

distribution

Results:

Mean peak bone marrow radioactivity level was about 37 ug/g at the 24-h time point, declining thereafter to about 10 ug/g by 72 h

Details on absorption:

not determined

Details on distribution in tissues:

Levels of radioactivity in blood were relatively constant between 2 and 48 h post-dose and then decreased slightly. Plasma levels were also relatively constant and higher than those in whole blood during the first 48 h, then decreased to whole blood levels (see Table 1). In the bone marrow, kidney and liver radioactivity levels increased to a maximum at between 24 and 32 h post-dose (see Table 1). In the testes, the radioactivity levels were generally lower than those in the blood (see Table 1).

Details on excretion:

not determined

Metabolites identified:

not measured

Table 1. Time course for distribution of test substance in tissues

	Mean ¿g test substance/g sample (n=3)
	2 h	4 h	8 h	15 h	24 h	32 h	48 h	56 h	72 h
Blood	6.8	8.6	6.1	6.9	6.9	7.6	6.5	4.4	4.4
Plasma	10	11	11	11	9	12	4.6	5.0	4.5
Testes	3.5	2.7	4.7	6.8	5.3	5.3	4.4	3.5	4.1
Kidneys	23	24	30	32	32	42	19	22	14
Liver	12	15	15	24	22	27	12	12	14
Bone marrow	7.3	6.1	8.9	22	37	21	11	11	10

Other findings: diarrhoea was observed in all animals at the 15- and 24-h necropsies, but was absent at 32 h onwards. Mean body weight had decreased by about 5% of the pre-dose weight by the 24-h necropsy but had returned to pre-dose weight at 72 h.

Conclusions:

In a study to determine whether orally administered trisodium EDDS reached the bone marrow, a single gavage dose (2160 mg/kg bw) of the radiolabelled test substance to male rats showed peak levels in liver, kidney, testes and bone marrow at between 15-32 h post-dose. In blood and plasma the levels were relatively constant during the first 48 h, before decreasing until study termination at 72 h.

Executive summary:

Trisodium EDDS was studied for its ability to reach the bone marrow under conditions similar to those used in in vivo cytogenetic assays.

Groups of three male rats per time point were administered a single dose of (14C-) radiolabelled trisodium EDDS by gavage at 2106 mg/kg bw (in water) and necropsied at 2, 4, 8, 15, 24, 32, 48, 56 or 72 h. Radioactivity was measured in the blood, plasma, testes, liver, kidneys and bone marrow (femur). Peak levels in liver, kidney, testes and bone marrow were found at between 15-32 h post-dose; the peak levels were 6.8, 42, 27 and 37 ug/g tissue for the testes, kidneys, liver and bone marrow, respectively. In blood and plasma, the levels were relatively constant at about 7 and 11 ug/g, respectively, during the first 32-48 h, before decreasing to about 4.5 ug/g at study termination..

In conclusion, radiolabelled trisodium EDDS was absorbed from the gastrointestinal tract to reach the blood, plasma, bone marrow, liver, kidney and testes before the levels declined from 48 h to study termination at 72 h. Results of this study demonstrate that bone marrow is exposed to EDDS and/or its metabolites following oral (gavage) dosing under conditions similar to those employed in in vivo cytogenetic studies.

Description of key information

In reliable studies, at least 5% of radiolabelled trisodium EDDS was considered absorbed after 72 h in male and female rats following single gavage administration and 11% following dermal application.

In the oral studies, trisodium EDDS was rapidly eliminated, mainly in the faeces (about 75% of the administered dose), whereas in the dermal studies less than 9% was excreted (the majority remained on the skin). Low amounts of radioactivity were detected in the organs and tissues examined in both studies (Ferinandi, 1995).

Good quality studies, involving single gavage administration of radiolabelled trisodium EDDS at about 2 g/kg bw to male (Powers, 1993a) and female (Powers, 1993b) rats, demonstrate that the bone marrow is exposed to EDDS and/or its metabolites following oral dosing under conditions similar to those employed in an in vivo cytogenetic study (Putman, 1994).

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

5

Absorption rate - dermal (%):

11

Additional information

In a GLP study conducted accorrding to a protocol similar to OECD Guideline 417, the absorption, distribution and elimination of 14C-labelled trisodium EDDS was determined in male and female Wistar rats. The radiolabelled test substance was administered by single oral gavage (in water) to five rats of each sex at a dose level of 0.5 mg/rat (about 2-3 mg/kg bw) and the animals placed in individual metabolism cages. Urine, faeces and cage washes were collected at 24, 48 and 72 h and expired air in 12, 24, 36, 48 and 72 h samples. At study termination (72 h), blood samples were taken and plasma separated and selected tissues and gastrointestinal contents were collected and the levels of radioactivity were determined by liquid scintillation counting (after solubilising the tissues as necessary). Trisodium EDDS was rapidly excreted, mainly in the faeces. In the first 24 h, approximately 62 and 70% of the administered dose, respectively in males and females, was excreted in the faeces, and after 72 h about 75% excreted in the faeces. At least 5% of the test substance was considered absorbed as determined by the amount of radioactivity in urine, expired air and in the tissues (additional material may have been collected in the cage washes and faeces). The combined mean radioactivity content of blood and tissues (including carcass) was 0.136 and 0.153% of the administered dose in males and females, respectively. No statistically significant gender differences were evident in the absorption, distribution or excretion patterns (Ferdinandi, 1995).

 

In a GLP study conducted according to a protocol similar to OECD Guideline 417, the absorption, distribution and elimination of 14C-trisodium EDDS was determined in male and female Wistar rats. The radiolabelled test substance was applied (in water) at 4-5 mg/kg bw in a glass chamber to the shaved skin of five rats of each sex for 72 h. Urine, faeces, cage washings and expired air were collected at intervals throughout the study period and the radioactive content was measured. At study termination, levels of radioactivity were determined in adipose tissue, brain, bone marrow, femur bone, gonads, heart, gastrointestinal tract, gastrointestinal contents, kidneys, liver, lungs, muscle, pancreas, spleen, carcass, skin from the treatment site and adjacent sites. The total recovery of radioactivity from all sources was 59.1% in males and 62.8% in females. This mainly represented unabsorbed material found at the skin treatment site (about 40%) and in the dermal chamber washings (about 10%). Only about 11% of the applied dose was considered absorbed in males and 5% in females during the 72-h exposure period, with less than 9% recovered in the excreta (urine, faeces, cage wash and expired air). Low amounts of radioactivity were detected in the organs and tissues examined, ranging from 0% to up to 1.2% in the male carcass (Ferdinandi, 1995).

 

In good-quality studies, trisodium EDDS was studied for its ability to reach the bone marrow in male (Powers, 1993a) and female (Powers, 1993b) rats, under conditions similar to those used in in vivo cytogenetic assays. Groups of three male rats per time point were administered a single dose of (14C-) radiolabelled trisodium EDDS by gavage at 2106 mg/kg bw (in water) and necropsied at 2, 4, 8, 15, 24, 32, 48, 56 or 72 h. Radioactivity was measured in the blood, plasma, testes, liver, kidneys and bone marrow (femur). Peak levels in liver, kidney, testes and bone marrow were found at between 15-32 h post-dose; the peak levels were 6.8, 42, 27 and 37 µg/g tissue for the testes, kidneys, liver and bone marrow, respectively. In blood and plasma, the levels were relatively constant at about 7 and 11 µg/g, respectively, during the first 32-48 h, before decreasing to about 4.5 µg/g at study termination (Powers, 1993a). Groups of three female rats per time point were administered a single dose of (14C-) radiolabelled trisodium EDDS by gavage at 2053 mg/kg bw (in water) and necropsied at 2, 8, 15, 25, 32, 37, 48, 56 and 72 h post-dose. Radioactivity was measured in the blood, plasma, liver, kidneys, ovaries and bone marrow (femur). Highest levels of radioactivity were found in the kidney and liver (peaking within 8 h post-dose). No distinct rise to peak radioactivity was seen in any of the tissues analysed. Plasma radioactivity was highest at 8 h post-dose, and blood and ovary radioactivity at 15 h post-treatment, and then slowly decreased. Liver, kidney and bone marrow levels were highest at 2, 8 and 24 h, respectively, and then were slowly reduced (Powers, 1993b). Results of these studies demonstrate that bone marrow is exposed to EDDS and/or its metabolites following oral (gavage) dosing under conditions similar to those employed in an in vivo cytogenetic study (Putman, 1994).