Pentasodium (carboxylatomethyl)iminobis(ethylenenitrilo)tetraacetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Standard Guideline study done according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The test substance is an irritant, the standard LLNA is known to provide a high number of false positive results for irritant substances.

Test material

Test material information

Constituent 1

Reference substance name:

N-carboxymethyliminobis(ethylenenitrilo)tetra(acetic acid)

EC Number:

200-652-8

EC Name:

N-carboxymethyliminobis(ethylenenitrilo)tetra(acetic acid)

Cas Number:

67-43-6

IUPAC Name:

N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine

Details on test material:

Diethylenetriaminepentaacetic acid
Lot no. QA2101D6F6
White Powder
99.41% purity

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Description, Identification and Housing:
Young adult, Hartley-derived albino guinea pigs were received on September 12, 2002, September 19, 2002 and September 26, 2002, from Hilltop Lab Animals, Inc., Scottdale, PA. Upon receipt, plastic ear tags displaying unique identification numbers were used to individually identify the animals. Cage cards displaying at least the study number, animal number and sex were affixed to each cage. The animals were housed individually in suspended stainless steel cages. All housing and care were based on the standards recommended by the Guide for the Care and Use of Laboratory Animals.
Environment:
The animal room temperature and relative humidity ranges were 61-74EF (16-23°C) and 31-80%, respectively. Environmental control equipment was monitored and adjusted as necessary to minimize fluctuations in the animal room environment. Light timers were set to maintain a 12-hour light/12-hour dark cycle and room ventilation was set to produce 10-15 air changes/hour. The room temperature and relative humidity were recorded a minimum of once daily.
Food:
PMI Certified Guinea Pig Chow #5026 (Purina Mills, Inc.) was provided ad libitum to the animals throughout the study. The lot number and expiration date of each batch of diet used during the study were recorded. The feed was analyzed and certified by the supplier for nutritional components and environmental contaminants. Dietary limitations for various environmental contaminants, including heavy metals, pesticides, polychlorinated biphenyls and total aflatoxin are set by the manufacturer. Within these limits, contaminants which may have been present were not expected to compromise the purpose of this study. Results of the dietary analyses (Certificates of Analysis) are provided by the manufacturer for each lot of diet. These are maintained by SLI.
Water:
Municipal tap water treated by reverse osmosis was available ad libitum throughout the study. The purified water was supplied by an automatic watering system. Monitoring of the drinking water for contaminants is conducted by SLI and the records are available for inspection. Within generally accepted limits, contaminants which may have been present were not expected to compromise the purpose of this study. The water meets the standards specified under the EPA National Drinking Water Regulations (40 CFR Part 141).
Acclimation:
Upon receipt, the animals were removed randomly from the shipping cartons, examined by qualified personnel, identified with plastic ear tags and then acclimated to the laboratory conditions for a minimum of five days. The animals were observed daily for overt physical or behavioral abnormalities, general health/moribundity and mortality.
Animal Selection:
The animals chosen for study use were arbitrarily selected from healthy stock animals to avoid potential bias. All animals received a detailed pretest observation prior to dosing. Only healthy animals were chosen for study use. Females were nulliparous and nonpregnant. For the range-finding study, the male animals were approximately 7-8 weeks of age and weighed 392-423 g prior to dosing and the female animals were approximately 9 weeks of age and weighed 380-421 g prior to dosing. For the main sensitization study, the male animals were approximately 7 weeks of age and weighed 365-419 g prior to Induction I and the female animals were approximately 8 weeks of age and weighed 333-406 g prior to Induction I.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Induction 2

Concentration / amount:

Following a range finding assay

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Challenge 2

Concentration / amount:

Following a range finding assay

No. of animals per dose:

10 males and 10 females.

Details on study design:

This study consisted of a topical range-finding group, a test group, a challenge control group [2]. A rechallenge control group was maintained on this study; however, the rechallenge procedure was not required since the challenge data was definitive.

Topical Range-Finding Study

Dosing:
On the day prior to dose administration, four topical range-finding guinea pigs were weighed and the hair removed from the right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures. On the following day, four concentrations of the test article were prepared and each concentration was applied to the clipped area of each topical range-finding animal as indicated below:

Concentration: 100%, test site: 1, amount applied: 0.25 g
Concentration: 75%, test site: 2, amount applied: 0.3 ml
Concentration: 50%, test site: 1, amount applied: 0.3 ml
Concentration: 25%, test site: 1, amount applied: 0.3 ml

For the diluted material the vehicle used was polyethylene glycol 400. For the 100% material, the test site was wetted using polyethylene glycol 400.

Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chambers and the animal was returned to its cage. Approximately six hours after chamber application, the binding materials were removed. The test sites were then wiped with gauze moistened with deionized water followed by dry gauze to remove test article residue. The animals were then returned to their cages.
Dermal observations:
The test sites of the r ange-finding animals were graded for irritation at approximately 24 and 48 hours following chamber application using the Dermal Grading System.
Clinical Observations:
Any unusual observations and mortality were recorded. The topical range-finding animals were observed for general health/mortality twice daily, once in the morning and once in the afternoon.
Body Weights:
Individual body weights were obtained for the topical range-finding animals on the day prior to dosing.
Scheduled Euthanasia:
Following the final scoring interval, all range-finding animals were euthanized by carbon dioxide inhalation. Gross necropsy examinations were not required for these animals.

Sensitization Study
Preliminary Procedures:
On the day prior to each dose administration, the guinea pigs had the hair removed with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
Dosing:
A dose of 0.25 g of the test article was placed in a 25 mm Hilltop chamber (with cotton pad removed) backed by adhesive tape (occlusive patch) and moistened with polyethylene glycol 400. The chambers were then applied to the clipped surface as quickly as possible. Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.
Induction:
On the day prior to the first induction dose administration (day -1), all test and control animals were weighed and the hair was removed from the left side of the test animals. On the day following clipping (day 0), chambers were applied as follows:

Day 1, Induction number 1: Concentration: 100%, test site 1, 10 males and 10 females
Day 7, Induction number 2: Concentration: 100%, test site 1, 10 males and 10 females
Day 14, Induction number 3: Concentration: 100%, test site 1, 10 males and 10 females

The induction procedure was repeated on study day 7 and on study day 14 so that a total of three consecutive induction exposures were made to the test animals.

Challenge:
On the day prior to challenge dose administration, the test and challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (day 28), chambers were applied as follows:

Group: Test
Test material: DTPA
Concentration 100%
Test site: 2
No. of animals: 10 male, 10 female

Group: Challenge control
Test Material: DTPA
Concentration: 100%
Test site: 2
No. of animals: 5 male, 5 female

Test Article Removal:
Approximately six hours after chamber application, the binding materials were removed. The test sites were wiped with gauze moistened in deionized water, followed by dry gauze, to remove test article residue. The animals were then returned to their cages.
Dermal Observations:
The test sites were graded for irritation at approximately 24 and 48 hours following chamber application (induction) or chamber removal (challenge) using the Dermal Grading System.
Clinical Observations:
Any unusual observations and mortality were recorded. The animals were observed for general health/mortality twice daily, once in the morning and once in the afternoon.
Body Weights:
Individual body weights were obtained for all sensitization study animals on the day prior to the first induction (day -1) and for the appropriate test and challenge control animals on the day prior to challenge dosing.
Scheduled Euthanasia:
All sensitization study animals were euthanized by carbon dioxide inhalation following each animal’s final scoring interval. Gross necropsy examinations were not required for these animals.

Protocol Deviations:
On several occasions, the animal room temperature and relative humidity ranges [61- 74°F (16-23°C) and 31-80%, respectively] were outside of the preferred ranges [63-73°F (17-23°C) and 30-70%, respectively] during this study. These occurrences were considered to have had no adverse effect on the outcome of this study.

Challenge controls:

Group: Challenge control
Test Material: DTPA
Concentration: 100%
Test site: 2
No. of animals: 5 male, 5 female

Positive control substance(s):

yes

Remarks:

1-chloro-2,4-dinitrobenzene and a-hexacinnamaldehyde. positive control data comes from historical control database of laboratory

Results and discussion

Positive control results:

Positive control historical database for the performing laboratory indicated that the assay is effective and reliable.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

some animals had evidence of dermal irritation outside of the test area.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: some animals had evidence of dermal irritation outside of the test area..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no signs of irritation

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no signs of irritation .

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

all animals demonstrated dermal irritation outside of the test material application site.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: all animals demonstrated dermal irritation outside of the test material application site..

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

one animal with irritation outside of the test site.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: one animal with irritation outside of the test site..

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Due to the complete lack of response in all animals challenged at 100% it is considered that DTPA is not a skin sensitiser under conditions of this study.

Executive summary:

The dermal sensitization potential DTPA was evaluated in Hartley-derived albino guinea pigs. Ten male and ten female guinea pigs were topically treated with 100% DTPA (0.25 g was moistened with six drops of polyethylene glycol 400), once per week, for three consecutive weeks. Following a two week rest period, a challenge was performed whereby the twenty test and ten previously untreated (naive) challenge control guinea pigs were topically treated with 100% DTPA (0.25 g was moistened with six drops of polyethylene glycol 400). Challenge responses in the test animals were compared with those of the challenge control animals. Following challenge with 100% DTPA (0.25 g was moistened with six drops of polyethylene glycol 400), dermal reactions in the test and challenge control animals were limited to scores of 0. Group mean dermal scores were noted to be the same in the test and challenge control animals therefore this substance was not considered to be a sensitiser under the conditions of this study.

DNCB: Using 1-Chloro-2,4-dinitrobenzene (DNCB) as a positive control, Springborn Laboratories, Inc., Spencerville, Ohio, has completed a study during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Modified Buehler Design). Following induction at 0.1% w/v DNCB in acetone/ethanol and challenge at levels of 0.1% and 0.05% w/v DNCB in acetone/ethanol, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent.

HCA: Using a-Hexylcinnamaldehyde (HCA) as a positive control, Springborn Laboratories, Inc., Spencerville, Ohio, has completed a study during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Modified Buehler Design). Following induction at 5% w/v HCA in ethanol and challenge at levels of 2.5% and 1% w/v HCA in acetone, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent. Based on the results of this study, XUS-40881.00 Experimental Chelating Agent (DTPA) is not considered to be a contact sensitizer in guinea pigs. The results of the DNCB and HCA historical control studies demonstrated that the test design utilized by Springborn Laboratories would detect potential contact sensitizers.