Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-08-15 until 1989-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according guideline 406

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

The skin sensitisation effect of tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate was examined in guinea pigs by a Guinea Pig Maximisation Test (GPMT) according to Magnusson and Kligman. The intradermal induction was done with 5% test item, followed by topical induction and challenge test, both with the undiluted (100%) test item (32.6% tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate in aqueous solution). The concentrations were chosen according to a pilot study. Before topical induction "pre-irritation" was induced by Sodium lauryl sulfate solution (10%).

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 1990. At this time an OECD guideline for a LLNA was not available.

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

EC Number:

266-442-3

EC Name:

Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Cas Number:

66669-53-2

Molecular formula:

C7H7Na4O9P

IUPAC Name:

tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Details on test material:

- The test item Bayhibit AM-tetra-sodium salt is composed of: 64.7% water, 32.6% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate, 1.3% Trisodium dihydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
- Molecular formula of the salt (if other than submission substance): C7H7O9PNa4
- Molecular weight of the salt: 358.1 g/mol.
- The test item is a clear liquid with a yellowish colour.
- Lot/batch number: 807168
- Kept in dark, 4°-10°C
- There was no need to check the stability of the test substance as the salt was produced nearly 4 months before the test was started.
- Homogeneity was visually stated.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: DHPW: Dunkin, Hartley, Peruvian, Winkelmann

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- SPF
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: Average 365 g. (315-405 g)
- Housing: cages Makrolon, type IV; 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Minimum of 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 50-60%
- Air changes (per hr): Around 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

water

Induction 2

Concentration / amount:

The doses for the induction and the challenge assay were selected according to a pilot study. For the induction exposure, the highest concentration that caused mild to moderate skin irritation was chosen and for the challenge exposure assay the highest non-irritant dose.
In the intradermal induction assay: 5% test item
In the topical induction assay: 100% test item
In the challenge assay: 100% test item

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Challenge 2

Concentration / amount:

The doses for the induction and the challenge assay were selected according to a pilot study. For the induction exposure, the highest concentration that caused mild to moderate skin irritation was chosen and for the challenge exposure assay the highest non-irritant dose.
In the intradermal induction assay: 5% test item
In the topical induction assay: 100% test item
In the challenge assay: 100% test item

No. of animals per dose:

In the treated group 20 guinea pigs.
2 control groups (one with, the other without challenge), in each group 10 guinea pigs.

Details on study design:

RANGE FINDING TESTS:
A pilot study was made. For the intradermal induction the 5% concentration was the highest examined with no observed reaction. For the topical induction and the challenge test the doses examination showed that the undiluted test item (32.6 % Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate in aqueous solution) had no skin reaction, 48 and 72 hours after each of the two tests.

MAIN STUDY
OVERVIEW ON TREATMENTS / EXPOSURES (test group, two control groups):
(1) Intradermal induction:
0.1 mL FCA and physiological NaCl solution, 1:1, 2*cranial;
0.1 mL test solution (5% test item in water), 2*medial;
0.1 mL FCM and test solution (FMC and 5% test item in water), 2*caudal:

(2) Topical induction with 0.5 mL test item (undiluted) for 48 hours after "pre-irritation" with Sodium lauryl sulfate (Dodecyl sodium sulfate) solution (10%) which makes the skin swelling and more easily affected.

(3) Challenge exposure:
Three weeks after the intradermal induction the animals were exposed to 0.5 mL test substance for 24 hours.

Control groups:
First control group: Intradermal induction (FCA and physiological NaCl solution, 1:1), the four other injections (2*medial, 2*caudal) without the test substance. No topical induction but "pre-irritation". Challenge with 0.5 mL test substance for 24 hours.
Second control group: Intradermal induction (FCA and physiological NaCl solution, 1:1), the four other injections (2*medial, 2*caudal) without the test substance. No topical induction but "pre-irritation". No challenge.


A. INDUCTION EXPOSURE (IN DETAIL)
- No. of exposures: 2 (intradermal induction followed by a topical induction)
- Exposure period: Day 0- the intradermal induction (first exposure), at day 7 after the intradermal induction an exposure by topical exposure.
- Test groups: 1 test group, 20 guinea pigs.
- Control groups: Two, 10 guinea pigs each.
The second control group is in the study for an optional second challenge test for that case that the result of the first challenge is not clear or the verify the dose-response relation if the results are positive.
- Site: Test area is cleared of hair. The 3 different injections (in the intradermal test ) are given each at the back, behind the neck in the right and in the left side of the spine. The gap between the injection location was 1-2 cm.
- Frequency of applications: One time injection in the intradermal test and in the following topical test a plaster with the undiluted test substance and a plaster with no test substance in the control group.
- Duration: Plasters (as described above) for 48 hours
- Concentrations: In the intradermal test :
Injection 1- a mixture (1:1) of FCA (Freunds Complete Adjuvant) and physiological saline.
Injection 2 - Test item 5% in water.
Injection 3 - A mixture (1:1) of test item in water with FCA.
In the control group same as written in the injection above but without the test item(instead the vehicle-water).

In the topical induction: plaster with undiluted (100%) test item. In the control groups plaster without the test substance.

B. CHALLENGE EXPOSURE (IN DETAIL)
- No. of exposures: 1 time, one plaster.
- Day(s) of challenge: The challenge test started 3 weeks after the intradermal injections.
- Exposure period: The plaster (with the undiluted test substance) was fixated and left on the skin for 24 hours.
- Test groups: 1 test group - 20 guinea pigs that already passed the intradermal and topical treatments.
- Control group: 1 control group - 10 guinea pigs that were not treated with the test item in the induction treatments were exposed in the challenge test to the test item the same as the treated group. The second control group did not receive a challenge dose.
- Site: In the treated and control group: On the left shoulder a plaster with the undiluted test item and on the right shoulder plaster without the test item.
- Concentrations: 0.5 mL of undiluted test item on a plaster.
- Evaluation (hr after challenge): 48 and 72 hours.

OTHER INFORMATION ON STUDY DESIGN:
- The bodyweights of all involved test animals were recorded before the test started at day 0 and at day 24.
- During the whole test the animals were observed for clinical symptoms (at least once a day).

Challenge controls:

Challenge with 0.5 mL test substance for 24 hours.

Positive control substance(s):

yes

Remarks:

The suitability of the Guinea pig strain DHPW for skin sensitization tests was examined with 1-chloro-2,4-dinitrobenzene. Diesing (Bayer report no. 18132 from 1989-06-26).

Results and discussion

Positive control results:

NA

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% (test item undiluted)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% (test item undiluted). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: negative control but challenged. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 4

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: negative control but challenged. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.5%

No. with + reactions:

12

Total no. in group:

12

Remarks on result:

other: The suitability of the Guinea pig strain DHPW for skin sensitization tests was examined with 1-chloro-2,4-dinitrobenzene. Diesing (Bayer report no. 18132 from 1989-06-26).

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test item caused no skin sensitisation effects.

Executive summary:

The skin sensitisation effect of tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate was examined in guinea pigs by a Guinea Pig Maximisation Test (GPMT) according to Magnusson and Kligman. The intradermal induction was done with 5% test item, followed by topical induction and challenge test, both with the undiluted (100%) test item (32.6 % Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate in aqueous solution). The concentrations were chosen according to a pilot study. Before topical induction "pre-irritation" was induced by Sodium lauryl sulfate solution (10%). No skin reaction was observed in the treated group after the challenge test compared to the controls. Moreover, the test animals' body weight development was examined and they were observed for clinical symptoms. No difference was exhibited between the treated and the control groups. Therefore, it can be concluded that under the test conditions, the test substance tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate, is not a potential skin sensitiser.