Trizinc dicitrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2-29 November 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to the appropriate OECD test guideline and very largely in compliance with GLP. The study is read across from trisodium citrate (CAS 68-04-2).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Exsting study from 1995

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium citrate

EC Number:

200-675-3

EC Name:

Trisodium citrate

Cas Number:

68-04-2

Molecular formula:

C6H8O7.3Na

IUPAC Name:

trisodium citrate

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Himalayan Spotted (Ibm:GOHI)

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd, Wölferstrasse 4, 4414 Fűllinsdorf/Switzerland
- Age at study initiation: 5-7 wk
- Weight at study initiation: 311-452 g (start of acclimatization)
- Housing: 1/Makrolon type 3 cage, autoclaved standard softwood bedding
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 1 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 50-74 (OECD guideline recommends 30-70%)
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 1994-11-02 To: 1994-11-29

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

water

Induction 2

Concentration / amount:

Induction: intradermal injections 5% TS (with Freund's Complete Adjuvant in some cases); epicutaneous 10% SLS followed by 75% TS.
Challenge: 75, 50 & 25% TS.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Challenge 2

Concentration / amount:

Induction: intradermal injections 5% TS (with Freund's Complete Adjuvant in some cases); epicutaneous 10% SLS followed by 75% TS.
Challenge: 75, 50 & 25% TS.

No. of animals per dose:

20 test animals, 10 vehicle controls, 2x10 positive controls

Details on study design:

RANGE FINDING TESTS:
Intradermal tests with 2 animals: 0.1 ml/site; 1, 3 and 5% TS in bidistilled water; evaluated at 24 h. 5% TS was selected
Epicutaneous tests with 4 animals: 2 cm x 2 cm saturated with 75, 50, 25 or 15% solutions of TS in bidistilled water, covered with aluminium foil, elastic plaster and impervious adhesive tape; 24-h contact; evaluation 24 h and 48 h after removal of dressing. 75% TS was selected for induction and challenge, and 50% and 25% were also selected for challenge.
The highest concentration tested, 75%, "was found to be the most qualified to assure an optimum technical application procedure" - it is not clear what this means

MAIN STUDY
A. INDUCTION EXPOSURE
intradermal injections (day 1)
- No. of exposures: 3 pairs of injections (0.1 ml/site)
- Test group: 1:1 Freund's Complete Adjuvant (FCA) and physiological saline; 5% TS in bidistilled water; 5% TS in 1:1 FCA: physiological saline
- Control group: 1:1 FCA and physiological saline; bidistilled water; 1:1 bidistilled water and 1:1 FCA: physiological saline
- Site: within a 4x6 cm clipped area

day 7: 6x8 cm area clipped. As no irritation had been observed in the pretest this area was pretreated with 10% sodium lauryl sulfate (SLS) in paraffinum per liquidum to provoke a mild inflammatory reaction.

epidermal applications (day 8, scapular area)
- No. of exposures: 1 (2x4 cm saturated filter paper)
- Exposure period: 48 h
- Test group: 2x4 filter paper saturated with 75% TS in bidistilled water, covered with aluminium foil, elastic plaster and impervious adhesive tape
- Control group: as for test group using bidistilled water
- evaluation at 24 and 48 h following removal of dressing

B. CHALLENGE EXPOSURE (day 22, flank)
Flanks shaved
- No. of exposures: 4 concentrations, each applied on saturated 2x2 cm filter paper
- Exposure period: 24 h
- Test groups: 75%, 50%, 25% and 0% TS in bidistilled water
- Control group: 75%, 50%, 25% and 0% TS in bidistilled water
- Site: left and right, caudal and cranial flanks
- Concentrations: 75%, 50%, 25% and 0% TS in bidistilled water
- Evaluation (hr after challenge): 24 h and 48 h

Challenge controls:

Naive controls: group of 10 males treated as described above.

Positive control substance(s):

yes

Remarks:

4-aminobenzoic acid ethyl ester (25 May to 27 June 1994); 2-mercaptobenzothiazol (25 May to 5 July 1994). Groups of 10 in each case.

Study design: in vivo (LLNA)

Statistics:

Mean and standard deviation for body weights.

Results and discussion

Positive control results:

Tests in the same strain of guinea pig using 4-aminobenzoic acid ethyl ester (25 May to 27 June 1994) and 2-mercaptobenzothiazol (25 May to 5 July 1994) (groups of 10 test and 4 or 5 controls; 25% in mineral oil in each case) found sensitization in 60% test animals and in none of the controls for both studies.

In vivo (non-LLNA)

Results

Reading:

other: readings at 24 and 48 h after challenge

Group:

test chemical

Dose level:

25, 50 and 75% TS

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No clinical signs or effect on body weight detected

Remarks on result:

other: Reading: other: readings at 24 and 48 h after challenge. Group: test group. Dose level: 25, 50 and 75% TS. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs or effect on body weight detected.

Any other information on results incl. tables

Table 1: Incidence of dermal response to challenge dosing

GROUP	MATERIAL	INTERVAL (AFTER 24 h CHALLENGE)	SKIN REACTIONS (NUMBERS OF ANIMALS)	NO. ANIMALS	SENSITIZATION INCIDENCE INDEX*
Test	TS (75%)	24 h	0	20	0/20=0%
		48 h	0	20
	TS (50%)	24 h	0	20
		48 h	0	20
	TS (25%)	24 h	0	20
		48 h	0	20
	Vehicle (bidistilled water)	24 h	0	20	0/20=0%
		48 h	0	20
Positive control (2-mercaptobenzothiazol)	25% in vehicle	24 h	6	10	6/10=60%*
		48 h	6	10
	Vehicle (mineral oil)	24 h	0	10	0/10
		48 h	0	10
Positive controls (4-aminobenzoic acid ethyl ester)	25% in vehicle	24 h	6	10	6/10=60%*
		48 h	6	10
	Vehicle (mineral oil)	24h	0	10	0/10=0%
		48h	0	10
Negative (vehicle) control	TS (75%)	24 h	0	10	n/a
		48 h	0	10
	TS (50%)	24 h	0	10
		48 h	0	10
	TS (25%)	24 h	0	10
		48 h	0	10
	Vehicle (bidistilled water)	24 h	0	10	n/a
		48 h	0	10

* A response of 30% or greater would indicate that a substance was a sensitizer

**The response of 60% in the positive controls confirms the sensitivity and reliability of the experimental technique

Applicant's summary and conclusion

Conclusions:

A well conducted, well reported guinea-pig maximization test failed to identify any sensitizing potential in tests involving a 75% concentration of the test material. The result is read across from trisodium citrate (CAS 68-04-2).