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Toxicological information

Skin sensitisation

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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:
1990-03-06 to 1990-04-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is well documented and performed according to generally valid and/or internationally accepted testing guidelines. The test material contained a high percentage of the test substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
Brake Fluid DOT 4 Super containing B-TTEGME
IUPAC Name:
Brake Fluid DOT 4 Super containing B-TTEGME
Details on test material:
- Name of test material (as cited in study report): Brake fluid DOT 4 Super
- Substance type: Borated Glycol Ether
- Physical state: Clear colourless liquid
- Analytical purity: 76% B-TTEGME
- Impurities (identity and concentrations): Confidential details on test material
- Composition of test material, percentage of components: Confidential details on test material
- Isomers composition: Not provided
- Purity test date: Not provided
- Lot/batch No.: KSLA Ref. 7842/89 (0.0039); Toxicology ref. No. ST90/023
- Expiration date of the lot/batch: Not provided
- Stability under test conditions: Not provided

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Porcellus.
- Age at study initiation: 5-9 weeks
- Weight at study initiation: 247 - 335 g
- Housing: steel cages with wire-mesh floors, 54 x 31 x 36 cm (2-3 animals/cage)
- Diet: pelleted diet (SG1 with vitamin C supplement, Grain Harvesters Ltd.) ad libitum
- Water: from the public supply was provided ad libitum
- Acclimation period: min. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%):30-70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1990-03-06 to 1990-04-06

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
RANGE FINDING TEST: 0.1 ml of several dilutions of the test material (0.06, 0.2, 0.6, 2.0% in water) for intradermal induction; 0.3 ml of several dilutions of the test material (10, 25, 60, 100% in water) for topical induction.
MAIN STUDY: 0.1 ml (0.6% in water) for intradermal induction; 0.3 ml undilted test material for topical induction; Challenge with 60% of test material.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
RANGE FINDING TEST: 0.1 ml of several dilutions of the test material (0.06, 0.2, 0.6, 2.0% in water) for intradermal induction; 0.3 ml of several dilutions of the test material (10, 25, 60, 100% in water) for topical induction.
MAIN STUDY: 0.1 ml (0.6% in water) for intradermal induction; 0.3 ml undilted test material for topical induction; Challenge with 60% of test material.
No. of animals per dose:
RANGE FINDING TEST: 2 male & 2 female guinea-pigs/dose group
MAIN STUDY: 10 male and 10 female guinea-pigs (5 male & 5 female guinea-pigs for controls)
Details on study design:
RANGE FINDING TEST:
2 male & 2 female guinea-pigs were closely shorn in the shoulder region using electric clippers followed by an electric razor. 0.1 ml of several dilutions of the test material (0.06, 0.2, 0.6, 2.0%) were injected intradermally on each side of the mid-line. The animals were examined on the following day to determine the maximum concentration that could be used in the main test without causing untoward toxicity.
The flank of each animal in further groups of 2 male and 2 female guinea-pigs, was further shorn. 0.3 ml dosis of several dilutions of the test material (10, 25, 60, 100%) were absorbed onto 16 cm2 Whatman No. 3 filter paper patches. The patches were applied to skin on the shorn flanks, covered by occlusive tape, and retained by elastic adhesive bandage for 24 hours. After removal of the patches and bandages the dermal test sites were examined for signs of irritation which were scored using a four point scale. The concentration selected for topical induction in the main test was that which just caused irritation and the concentration chosen for topical challenge was that which was just non-irritant.
MAIN STUDY:
A. INDUCTION EXPOSURE
The animals were closely shorn in the shoulder region using electric clippers followed by an electric razor: two rows of intradermal injections were made, one of either side of the mid-line, as follows:
test animals: anterior sites (0.1 ml of FCA); middle sites (0.1 ml of test material 0.6% in vehicle); posterior sites (0.1 ml of test material0.6% in 50:50 FCA/vehicle)
control animals: anterior sites (0.1 ml of FCA); middle sites (0.1 ml of vehicle); posterior sites (0.1 ml of 50:50 FCA/vehicle)
Freunds Complete Adjuvans (FCA) was prepared for use as a 50% v/v aqueous emulsion. One week after induction by intradermal injection, the same area of dorsal skin was shaven using electric clippers only. A 16 cm2 patch of Whatman No. 3 filter paper was moistened with 0.3 ml of the undiluted test material and placed over the sites of intradermal injections. The patches were covered with occlusive tape and held in place by elastic adhesive bandage for 48 hours. Similar patches of filter paper moistened with the vehicle alone were applied to the control group guinea-pigs. Any abnormal reactions to the induction procedure were recorded.
B. CHALLENGE EXPOSURE
Challenge was carried out 3 weeks after the intradermal induction. Hair was removed from one flank of all test and control animals by clipping and shaving. A 4 cm2 patch of Whatman No. 3 filter paper, moistened with 0.1 ml of 60% of test material, was placed on the shaven area, covered by occlusive tape and held in position by elastic adhesive bandage. After 24 hours the patches and bandages were removed and the challenge sites examined for any response. The response was scored using a four point scale. The result of the test is expressed as the number of positive responses shown by the test animals at 24 and/or 48 hours after removal of the challenge patches. the frequency of positive responses rather than their intensity is regarded as the important statistic in this test.

SCORING SYSTEM:
4 point scale used:
0 = No difference from surrounding skin
1 = Slight redness, edges not defined
2 = Pink/red area with defined edges
3 = Beet red area with well defined edges
OTHER: Individual body weights were recorded at the beginning of the main study and before challenge.
Challenge controls:
60% of test material
Positive control substance(s):
no

Study design: in vivo (LLNA)

Vehicle:
not specified
Concentration:
Not applicable (The Maximisation assay according to Magnussen and Kligman was conducted)
Details on study design:
Not applicable
Statistics:
Not applicable

Results and discussion

Positive control results:
Not applicable

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
60 % brake fluid (46% B-TTEGME)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 0.0. Group: test group. Dose level: 60 % brake fluid (46% B-TTEGME). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
0
Group:
negative control
Dose level:
60 % brake fluid (462% B-TTEGME)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 0.0. Group: negative control. Dose level: 60 % brake fluid (462% B-TTEGME). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
60 % brake fluid (46% B-TTEGME)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 60 % brake fluid (46% B-TTEGME). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
60 % brake fluid (46% B-TTEGME)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 60 % brake fluid (46% B-TTEGME). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
60 % brake fluid (46% B-TTEGME)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 60 % brake fluid (46% B-TTEGME). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
other: 3th reading
Hours after challenge:
48
Group:
negative control
Dose level:
60 % brake fluid (46% B-TTEGME)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: other: 3th reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 60 % brake fluid (46% B-TTEGME). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

The detailed results of the maximization test are provided here.

Table 1. Respons after intradermal induction (dose range finding)

Animal No.

Sex

0.06%

0.2%

0.6%

2.0%

321

M

1

1

1

2

322

M

1

1

1n

2n

301

F

1

1

1

2n

303

F

1

1

1n

2n

n – slight necrosis at injection site

 

Table 2. Respons after topical induction (dose range finding)

Animal No.

Sex

10% conc.

25% conc.

323

M

0

0

324

M

0

0

304

F

0

0

305

F

0.

0

 

Animal No.

Sex

60% conc.

Undiluted

325

M

0

0

326

M

0

1

307

F

0

0

308

F

0.

1

 


Table 3. Respons after intradermal/topical induction & topical challenge (main study)

Group

Animal No.

Sex

Body weight (g)

Response to challenge at time

Start

End

0h

24h

48h

Test animals

371

M

521

678

0

0

0

372

M

540

684

0

0

0

373

M

508

665

0

0

0

374

M

520

657

0

0

0

375

M

510

720

0

0

0

376

M

473

715

0

0

0

377

M

539

703

0

0

0

378

M

540

670

0

0

0

379

M

545

720

0

0

0

380

M

560

740

0

0

0

354

F

412

562

0

0

0

355

F

498

507

0

0

0

356

F

412

615

0

0

0

357

F

430

560

0

0

0

358

F

480

575

0

0

0

381

F

378

516

0

0

0

382

F

412

560

0

0

0

383

F

400

541

0

0

0

384

F

452

533

0

0

0

385

F

412

605

0

0

0

Control animals

391

M

565

795

0

0

0

392

M

618

812

0

0

0

393

M

548

765

0

0

0

394

M

535

740

0

0

0

395

M

536

720

0

0

0

386

F

418

652

0

0

0

387

F

418

580

0

0

0

388

F

418

586

0

0

0

389

F

469

600

0

0

0

390

F

427

601

0

0

0

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In the guinea-pig maximization test of Magnussen and Kligman none of the twenty test animals and 10 control animals showed a positive respons 24 or 48 hours after removal of the challenge patches.
Executive summary:

Brake Fluid DOT 4 Super is considered to have a similar toxicological profile as B-TTEGME. Brake fluid DOT 4 Super (containing 76% B-TTEGME) was tested for sensitization in the guinea-pig maximization test of Magnussen and Kligman. In a dose range finding test in 2 male and 2 female guinea-pigs/group, 0.1 ml (0.6% in water) and 0.3 ml (undiluted test material) showed slight irritancy and were therefore selected for intradermal and topical induction for 48h, respectively. For challenge, 60% test material in water was applied under occlusive tape for 24h . None of the 10 male and 10 female test animals showed a positive respons 24 or 48 hours after removal of the challenge patches. None of the 5 male and 5 female control animals showed any response to application of the 60% test material in water.