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Description of key information

The derived skin sensitisation effects from the key study was:
No sensitising effects observed, according to OECD 406, Shell, 1990;

B-TEGME is considered not to be sensitising for skin based upon systematic negative findings in various Magnussen and Kligman sensitisation tests with brake fluids. Sensitisation was tested by epidermal and topical induction, followed by topical challenge under occlusion in guinea-pigs.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-03-06 to 1990-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
This study was performed before test guidance OECD 406 was finalised. However, the study was performed according to the test guideline requirements.
Deviations:
no
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
In accordance with Annex VII of EC legislation 1907/2006 in-vivo testing studies apart from murine local lymph node assay (LLNA) meet the regulatory requirements if they were carried out or initiated before 11 October 2016. The described experimental test was performed according to the OECD 406 (GPMT method or Buehler test method) and meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4).
Specific details on test material used for the study:
- Substance type: Borated Glycol Ether
- Physical state: Clear colourless liquid
- Analytical purity: 37 % B-TEGME (test material is a mixture)
- Purity test date: Not provided
- Lot/batch No.: KSLA Ref. 7842/89 (0.0039); Toxicology Ref. ST90/023
- Expiration date of the lot/batch: Not provided
- Stability under test conditions: Not provided
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
Route:
intradermal
Vehicle:
water
Concentration / amount:
0.06, 0.2, 0.6, 2.0% / 0.1 ml of several dilutions
Day(s)/duration:
24 h
Adequacy of induction:
other: Range finding study: determine the maximum concentration without toxicity
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
10, 25, 60, 100% / 0.3 mL of several dilutions
Day(s)/duration:
24 h
Adequacy of induction:
other: Range finding study: examination for signs of irritation
Route:
intradermal
Vehicle:
water
Concentration / amount:
0.6 % / 0.1 mL
Adequacy of induction:
other: concentration that caused no unwanted toxicity
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
undiluted test material / 0.3 mL
Day(s)/duration:
48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
10, 25, 60, 100% / 0.3 mL
Day(s)/duration:
24 h
Adequacy of challenge:
other: Range finding study: examination for signs of irritation
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
60 % / 0.1 mL
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
RANGE FINDING TEST: 2 male & 2 female guinea-pigs/dose group
MAIN STUDY: 10 male & 10 female guinea-pigs (5 male & 5 female guinea-pigs for controls)
Details on study design:
RANGE FINDING TEST:
1)
- 2 male & 2 female guinea-pigs
- 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%) injected intradermally on each side of the mid-line
- examination on the following day: determination of maximum concentration used in the main test without causing untoward toxicity
2)
- 2 male & 2 female guinea-pigs
- flank of each animal shorn as described before
- 0.3 ml dosis of several dilutions of the test material (10, 25, 60, 100%) absorbed onto 16 cm2 Whatman No. 3 filter paper patches and applied to skin on the shorn flanks
- coverage: occlusive tape, retained by elastic adhesive bandage for 24 hours
- examination after removal of patches and bandages for signs of irritation (scoring: four point scale as described below)
- topical induction concentration for the main test: concentration just causing irritation; topical challenge concentration: concentration just non-irritant

MAIN STUDY:
A. INDUCTION EXPOSURE
- 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 material 0.6% 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): prepared as a 50% v/v aqueous emulsion]
- one week after induction: same area of dorsal skin shaven using electric clippers only
- 16 cm2 patch of Whatman No. 3 filter paper moistened with 0.3 ml of the undiluted test material, placed over the sites of intradermal injections
- patches covered with occlusive tape and held in place by elastic adhesive bandage for 48 hours
- filter paper moistened with the vehicle alone and applied to the control group guinea-pigs
- any abnormal reaction to the induction procedure was recorded

B. CHALLENGE EXPOSURE
- carried out 3 weeks after the intradermal induction
- clipping and shaving used to remove 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:
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
Reading:
1st reading
Hours after challenge:
0
Group:
test group
Dose level:
60 % brake fluid (22% B-TEGME)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
0
Group:
negative control
Dose level:
60 % brake fluid (22% B-TEGME)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
60 % brake fluid (22% B-TEGME)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
60 % brake fluid (22% B-TEGME)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
48
Group:
test group
Dose level:
60 % brake fluid (22% B-TEGME)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3th reading
Hours after challenge:
48
Group:
negative control
Dose level:
60 % brake fluid (22% B-TEGME)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation

Table 1: Response 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: Response 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: Response 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

 

Interpretation of results:
GHS criteria not met
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 (containing 37% B-TEGME) was tested for sensitization in the guinea-pig maximization test of Magnussen and Kligman and according to the OECD 406. 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 (equivalent to 22% B-TEGME) in water was applied under occlusive tape for 24h.

None of the 10 male and 10 female test animals showed a positive response 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% (equivalent to 22% B-TEGME) test material in water.

It can be occluded that B-TEGME is not sensitizing to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No sensitisation was observed in three studies with Brake fluids containing B-TEGME according to the guinea-pig maximization test, which is accepted as a relevant method. Although no studies were available with pure B-TEGME, the studies were performed according to international accepted testing guidelines with appropriate concentrations that were based upon preliminary testing. The studies were consistent in approach and results, and were therefore considered to be adequate and reliable. Since this technique has been shown to detect substances of weak skin sensitizing potential, it is unlikely that B-TEGME would be a sensitizer in man.

The key study for skin sensitisation was performed with a brake fluid containing 37% B-TEGME, which was tested in the guinea-pig maximization test of Magnussen and Kligman (Shell, SBGR 90.189, 1990). In a dose range finding test in 2 male and 2 female guinea-pigs/group, 0.6% in water and undiluted test material were selected for intradermal and topical induction, respectively. For challenge, 60% test material in water was applied under occlusive tape for 24h . None of the animals showed a positive response at 24 and 48 hours after removal of the challenge patches. The study was conducted according to the guinea-pig maximization test, which is accepted as an older but relevant method. Since this Magnussen and Kligman technique has been shown to detect substances of weak skin sensitizing potential and the B-TEGME containing brake fluid was not sensitizing, it is unlikely that B-TEGME would be a sensitizer in man.

Secondly, brake fluid was also negative for skin sensitisation in 10 male and & 10 female guinea-pigs according to the guinea-pig maximization procedure, with 5% dilution in water for intradermal induction and undiluted test material for topical induction, followed by 50% dilution of test material in water for challenge (Shell, TLGR.0090.77, 1977). In another study with Brake fluid 500 DOT 4 there was also no skin sensitisation with 1% w/v dilution in corn oil and undiluted test material for topical induction and challenge (Shell, TLGR.0015.75, 1975). No reactions for sensitisation or irriation were observed in both studies in tested and control animals.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.