A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

Endpoint summary

Description of key information

Skin sensitisation - in vivo (Buehler test)

Not sensitising.

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:

January 28, 1992 to February 28, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Organization for Economic Cooperation and Development (OECD) Guidelines for testing of chemicals
(Section 4, Subpart 406, Paris 1981).

Deviations:

not specified

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Available study pre-dates LLNA method and is performed in accordance with GLP to international recognised guidelines.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Species and strain: Dunkin Hartley albino guinea pigs
Justification for the selection of the test system: Dunkin Hartley albino guinea pig is the generally recommended species by Health Authorities in experimental model for skin sensitization studies
Supplier: Charles River Italia S.p.A., Via Indipendenza, 11, 22050 Calco (Como)
Shipping slip no. 00684, dated January 24, 1992 (20 animals)
Shipping slip no. 08363, dated October 31, 1991 (6 animals, preliminary test)

Number:
26 animals
10 animals/group
6 animals for the preliminary test (performed on December 11, 1991)

Body weight (and age): between 370-433 grams at the start of the experiment (corresponding to 2-3 months old)
Sex: male

Acclimatization: five days. Animals were observed daily to ascertain their fitness for the study.
Housing: (room T12C)
2 or 3 animals/cage in air-conditioned room.
-temperature: 22°C 2 2
-ail- changes: about 20/hour filtered on HEPA 99.97%
-relative humidity: 55% + 10
-light: 12 hours cycle (7 a.m. - 7 p.m.)
-cage: wire cages (40.5x38.5x18h) with stainelss steel feeder.
The waste that drops through the wire bottom on a removable paper was periodically disposed of.
An automatic standby power supply was not brought into operation as the main supply did not fail.

Animal identification: by coloring different area of the ears. A computerized randomization program was adopted allocate the animals to groups.
Cage identification: by cage card giving indelible experiment number, start day, the group and the subgroup (1a, 1b; 2a, 2b) identification.

Diet: the animals were fed a breeder standard GLP diet -certificate coded 8 GP 22, produced by Charles River Italia's feed licensee Mucedola S.r.l., Settimo bfilanese.
The declared contents, on the label, on dry matter basis (moisture 12%), were:
crude protein 19%
crude fat 4%
crude fiber 14.5%
ash 7.5%
The diet was supplemented by the Producer with vitamins and trace elements. According to the analytical certificates provided by the Supplier, the contents of the batch of diet used in this study were within ± 5% of the declared values and contaminants were within the limits proposed by EPA-TSCA (44FR:44053-44093, July 26, 1979).
The animal feed, in compliance with RBM SOPS is analyzed twice a year for bacterial contamination.
The diet was available to the animals "ad libitum".

Water: from the municipal water main system. Water is filtered and distributed "ad libitum" to the animals by an automatic valve system.
Periodically drinking water is analyzed for microbiologic count, heavy metals, other contaminants (e.g. solvents, pesticides) and other chemical and physical characteristics.
The acceptable limits of quality of the drinking water are those defined in EEC Directive 80/778.
Contaminants that might interfere with the objectives of the study are not expected to be present in the diet or in the drinking water.
The analytical certificates of animal feed and water are filed at RBM premises.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

50% (0.5 ml)

Day(s)/duration:

Day 0/6 hours

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

25% (0.5 ml)

Day(s)/duration:

Day 0/6 hours

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.5 ml

Day(s)/duration:

Day 27/24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 animals in test group
10 animal in negative control group

Details on study design:

Test article dosage levels
As reported in the protocol the preliminary test was performed in order to select a non irritating test article concentration.
The test article was assayed by occlusive patch on two animals for each concentration.
Three doses undiluted, 50% and 25% were tested.
0.5 ml of each test article concentration was applied onto the skin with a closed patch.
24 hours after the administration, the patches were removed, the animals were observed up to 48 hours for reaction at the skin area of the patch application.
All three doses were well tolerated and therefore the test article undiluted was used in the sensitization test.

Test article formulates
The test article formulate was used undiluted.

Test description
Administration route: topical exposure by occlusive patch
Dose administered: the test article was used undiluted
Observation of clinical sign: daily, be cage
Bodyweight recording: pre-trial and weekly thereafter
Experimental group: 1 of 10 animals
Negative control group: 1 of 10 animals.
The animals of this group were treated only at day 28 with the test article closed patch as the product was tested undiluted.

Experiment design
Induction
Day -1: the animals of group 1 were clipped on the shoulder region in an area of about 6x6 cm with an electric clipper, before treatment.

Day 0: the animals clipped the day before were treated with the test article. 0.5 ml of the product was held on contact with the skin of the shoulder region by mean of an occlusive patch for 6 hrs.

Day 7 and 14: the 6 hrs closed-patch was repeated (see day 0).

Challenge
Day 27: in the animals of the experimental or negative control group one flank was clipped in an area of about 2x2 cm.

Day 28: the challenge test was identical to the occlusive patch test of the inductive phase, unless the patch was held in place for 24 hrs.

Day 29: the patch was removed, the area of challenge marked, and the skin reaction was read.

Day 30: a second reading was done.

Assessment of skin reactivity
Evaluation of skin reaction:
0 normal skin
1 slight erythema
2 moderate-intense erythema, slight edema
3 marked erythema and edema
The results were expressed in terms of incidence and severity of response:
incidence: the number of animals showing responses of 1 or greater at either 24 or 48 hrs divided by the number of animals tested;
severity: the sum of the test grades divided by the number of animals tested.

Challenge controls:

the challenge test was identical to the occlusive patch test of the inductive phase

Positive control substance(s):

not required

Positive control results:

Not required

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

 Skin sensitisation test in guinea pigs (Buhler test)

Bodyweight (grams)

Group no.	Guinea pig no.	Days
		-1	6	13	21	27
1	1 2 3 4 5 6 7 8 9 10	400 405 404 370 407 386 420 395 379 405	450 478 471 443 500 429 491 466 453 467	498 547 541 499 557 475 552 430 495 550	532 616 611 561 635 545 607 609 556 605	574 669 667 612 668 573 678 657 608 708
2	11 12 13 14 15 16 17 18 19 20	408 433 422 405 397 405 404 417 406 421	483 494 506 480 476 474 470 507 504 491	542 538 585 545 527 538 538 575 584 548	597 569 656 593 607 613 637 632 604 655	636 588 713 666 648 674 695 680 710 636

 

Skin sensitisation test in guinea pigs (Buhler test)

Experimental group No. 1

Substance: [ANOX BF]

	Challenge
Days	Test article 29	Test article 30
Guinea pig no.
1 2 3 4 5 6 7 8 9 10	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0

 

0: No reaction

1, 2, 3: skin reaction was assessed according to the scale described in the text. Animals showing reaction ≥ 1 were considered positive

No. of positive animals at the challenge: 0

Results: NEGATIVE

 

Skin sensitisation test in guinea pigs (Buhler test)

Experimental group No. 2

Substance: [ANOX BF]

	Challenge
Days	Test article 29	Test article 30
Guinea pig no.
11 12 13 14 15 16 17 18 19 20	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0

 

0: No reaction

No. of positive animals at the challenge: 0

Results: NOT IRRITANT

Interpretation of results:

GHS criteria not met

Conclusions:

The sensitizing potential of the test article [ANOX BF] was assessed in guinea pigs using the Buhler test as described by Klecak.
No animal treated with the test article showed positive reaction at the challenge.
On the basis of these results, under the experimental conditions applied, [ANOX BF] did not appear to possess sensitizing capacity.

Executive summary:

The sensitizing potential of the test article [ANOX BF] was assessed in guinea pigs using the Buhler test as described by Klecak.

In the group treated with the test article no animals showed positive reaction at the challenge.

Under the experimental conditions applied, [ANOX BF] did not appear to possess sensitizing properties.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation - in vitro (Buehler test)

In the group treated with the test article no animals showed positive reaction at the challenge.

Under the experimental conditions applied, [ANOX BF] did not appear to possess sensitizing properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available Buehler skin sensitisation study, the substance does not meet the classification criteria for skin sensitisation in accordance with Regulation 1272/2008.