Registration Dossier

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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- The test substance 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 substance 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)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
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% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
In the topical induction assay: 100% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
In the challenge assay: 100% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
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% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
In the topical induction assay: 100% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
In the challenge assay: 100% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate
No. of animals per dose:
In the treated group 20 guinea pigs.
2 control groups (one with, the other without challenge), in each one 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 substance (100% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate) 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% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate in water), 2*medial;
0.1 mL FCM and test solution (FMC and 5% Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate in water), 2*caudal:

(2) Topical induction with 0.5 mL test substance (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 - Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate 5% in water.
Injection 3 - A mixture (1:1) of Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate in water with FCA.
In the control group same as written in the injection above but without the test substance (instead the vehicle-water).

In the topical induction: plaster with undiluted (100%) Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate. 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 substance in the induction treatments were exposed in the challenge test to the test substance 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 substance and on the right shoulder plaster without the test substance.
- Concentrations: 0.5 mL of undiluted test substance 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):
no

Study design: in vivo (LLNA)

Concentration:
NA
No. of animals per dose:
NA
Details on study design:
NA
Statistics:
NA

Results and discussion

Positive control results:
NA

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
100% (test substance 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 substance undiluted). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
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.
Reading:
1st reading
Hours after challenge:
48
Group:
other: negative control but challenged
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:
Reading:
2nd reading
Hours after challenge:
72
Group:
other: negative control but challenged
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:

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: NA
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: NA

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the study conditions, the test substance 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 substance, followed by topical induction and challenge test, both with the undiluted (100%) test substance. 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.