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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2000-05-23 - 2000-06-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented GLP study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Gefahrstoffverordnung (GefStoffV) of 29 September 1994 (BGBI. I, p. 2557)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EEC Directive (67/548/EEC), 7th Amended Directive (92/32/EEC) & EEC Directive 93/21/EEC
Deviations:
not specified
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
other: Pirbright white
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: male: 361 - 439 g; female: 300 - 338 g (main test)
- Housing: Before the animals arrived, the study room and cages were cleaned and disinfected. During the study, the room and cages were cleaned at regular intervals. The guinea pigs were housed collectively up to a maximum of 5 animals per cage in a battery of cages, equipped with a paper disposal system.
- Diet (e.g. ad libitum): Pelleted diet, offered ad libitum.
- Water (e.g. ad libitum): Tap water as for human consumption was continuously available ad libitum via drinking nipples.
- Acclimation period: 6 days (range finding); 8 days (main test)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3° C (monitored twice daily except weekends, when they were monitored once a day)
- Humidity (%): between 30 and 70 % (monitored twice daily except weekends, when they were monitored once a day)
- Air changes (per hr): Air was changed about 16 times per hour and filtered adequately.
- Photoperiod (hrs dark / hrs light): Artificial light was set to give a cycle of 12 hours light and 12 hours dark with light on at 7.00 a.m.
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal: aqua ad iniectabilia and Freund's complete adjuvant (FCA); epicutaneous: undiluted
Concentration / amount:
For the intradermal injection, the test article was diluted with the vehicle to a final concentration of 5 %. For the dermal application, the test article was used undiluted.
Route:
epicutaneous, occlusive
Vehicle:
other: intradermal: aqua ad iniectabilia and Freund's complete adjuvant (FCA); epicutaneous: undiluted
Concentration / amount:
For the intradermal injection, the test article was diluted with the vehicle to a final concentration of 5 %. For the dermal application, the test article was used undiluted.
No. of animals per dose:
Two animals (1 male, 1 female) were used in the preliminary test, 10 test (5 males and 5 females) and 5 control animals (3 males and 2 females) were employed in the main test.
Details on study design:
RANGE FINDING TESTS:
The range finding test was performed to determine the concentrations of the test article to be used in the main test.
For the intradermal injection, the test article was diluted with aqua ad iniectabilia and Freund's complete adjuvant (A.a.i.; FCA) to a final concentration of 5 %. Two animals were employed, skin reactions were recorded 48 h after treatment. For the dermal application, the test article was used undiluted. A closed patch exposure was effected by means of an occlusive bandage and non-irritating tape, which enveloped the whole animal's trunk. Two animals were employed and skin reactions were recorded 48 h post applicationem.

MAIN STUDY:
The main study was performed on 10 test and 5 control animals. On the basis of the results of the range finding test, the concentration of 5 % of the test article was considered to be suitable for intradermal injection and the undiluted test article for dermal application.
A. INDUCTION EXPOSURE
A.1 FIRST STAGE - In each test animal, three pairs of intradermal injections (0.1 mL) are made in the clipped intrascapular region on either side of the spine
- Test groups:
1) FCA 50 % (v/v) diluted in aqua ad iniectabilia,
2) test article 5 % in aqua ad iniectabilia,
3) test article 5 % in aqua ad iniectabilia/FCA.
- Control group:
1) FCA 50 % (v/v) diluted in aqua ad iniectabilia,
2) aqua ad iniectabilia,
3) aqua ad iniectabilia 50 % (v/v) diluted in FCA.
A.2 SECOND STAGE - Seven days later, the previous injection sites were covered occlusively for 48 h with a patch carrying the test article or, in control animals, the vehicle. Since the undiluted test article was non-irritating after dermal application in the range finding test, the skin area was reclipped and pretreated with 10 % sodium lauryl sulfate in vaseline 24 h before application of the test article to induce a mild inflammation.

B. CHALLENGE EXPOSURE
Both control and test animals were subjected to a challenge exposure 14 days after the second stage of the induction. The challenge test was performed on a 5 x 5 cm clipped skin area on each flank. The test article was applied to the left flank and the vehicle aqua ad iniectabilia to the right in a volume of 0.5 ml. In each case, the duration of the exposure was 24 h under an occlusive dressing.
Challenge controls:
The vehicle aqua ad iniectabilia was applied to the right flank as control.
Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester (benzocaine)
Positive control results:
According to the maximisation scheme of Magnusson and Kligman (J. Invest. Dermatol., 52, 268-276, 1969), the positive control article
"4-Aminobenzoic acid ethyl ester (benzocaine)" is classified as a "moderate sensitizer" at the concentration of 25 %.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
test article concentration: 100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: test article concentration: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
test article concentration: 100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: test article concentration: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Range Finding Test

Intradermal injection - No skin reactions were observed after the intradermal injection of the test article at a concentration of 5 %.

Table 1: Individual observations of skin reactions in the range finding test 48 h after intradermal injection

 Animal No.  Sex  Body weight (g)  Concentration (%)  Skin reactions
 1  m  354  5  no specific findings
 2  f  302  5  no specific findings

Dermal application - No skin reactions were observed after the dermal application of the undiluted test article.

Table 2: Individual observations of skin reactions in the range finding test 48 h after dermal application

Animal No.   Sex  Body weight (g)  Concentration (%)  E  Oe
 1  m  354  100  0  0
 2  f  302  100  0  0

m = male; f = female; E = erythema; Oe = oedema

Main Test

There were no skin reactions after the dermal application in the induction phase and after challenge exposure. The sensitisation rate, i.e. the number of animals showing an allergic response expressed as a percentage of the total number of animals, was determined at 24 and 48 h after the patch removal and was 0 % in each case.

Table 3: Individual values of responses in the main test after challenge exposure in the test group (test article concentration: 100 %)

Animal      Initial BW  24 h           48 h           Final BW
 No.  Sex    Test article     Control article     Test article     Control article     
     (g)  E  Oe  E  Oe  E  Oe  E  Oe  (g)
 1  m  397  0  0  0  0  0  0  0  0  565
 2  m  421  0  0  0  0  0  0  0  0  659
 3  m  384  0  0  0  0  0  0  0  0  574
 4  m  361  0  0  0  0  0  0  0  0  504
 5  m  392  0  0  0  0  0  0  0  0  620
 6  f  327  0  0  0  0  0  0  0  0  491
 7  f  345  0  0  0  0  0  0  0  0  497
 8  f  338  0  0  0  0  0  0  0  519
 9  f  310  0  0  0  0  0  0  0  0  458
 10  f  300  0  0  0  0  0  0  0  0  404

Reaction rate at 24 h and at 48 h: 0 %; m = male; f = female; E = erythema; Oe = oedema

Table 4: Individual values of responses in the main test after challenge exposure in the control group (test article concentration: 100 %)

 Animal     Initial BW  24 h           48 h           Final BW
 No.  Sex    Test article   Control article     Test article     Control article     
     (g)  E  Oe  E  Oe  E  Oe  E  Oe  (g)
 1  m  371  0  0  0  0  0  0  0  0  565
 m  439  0  0  0  0  0  0  0  0  574
 3  m  386  0  0  0  0  0  0  0  0  574
 4  f  308  0  0  0  0  0  0  0  0  478
 5  f  312  0  0  0  0  0  0  0  0  414

Reaction rate at 24 h and at 48 h: 0 %; m = male; f = female; BW = body weight; E = erythema; Oe = oedema

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS
Conclusions:
There were no skin reactions after the dermal application in the induction phase and after challenge exposure. The sensitisation rate, i.e. the number of animals showing an allergic response expressed as a percentage of the total number of animals, was determined at 24 and 48 h after the patch removal and was 0 % in each case.
Executive summary:
The potential skin sensitising properties of the test article acetyltri-2 -ethylhexylcitrate (ATEHC, CAS 144-15-0) were assessed in a guinea pig maximisation sensitisation test according to OECD 406 and EU method B.6 and in compliance with GLP (2000) using 10 test and 5 control animals. Following the induction exposure to the test article or the vehicle (control article) aqua ad iniectabilia, the animals of both groups were subjected two weeks later to a challenge exposure with the test article and the control article. Responses to the challenge procedure were evaluated 24 and 48 h after the end of the exposure period. No allergic skin reactions were observed at 24 and 48 h after the end of the challenge procedure; the sensitisation rate was 0 %.

It can be assumed that the same applies to tributyl citrate (CAS 77-94-1) as it is a near analogue to the test substance acetyltri-2 -ethylhexylcitrate (ATEHC).

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

The potential skin sensitising properties of the test article acetyltri-2 -ethylhexylcitrate (ATEHC, CAS 144-15-0) were assessed in a guinea pig maximisation sensitisation test according to OECD 406 and EU method B.6 and in compliance with GLP (Bien, 2000) using 10 test and 5 control animals. Following the induction exposure to the test article or the vehicle (control article) aqua ad iniectabilia, the animals of both groups were subjected two weeks later to a challenge exposure with the test article and the control article. Responses to the challenge procedure were evaluated 24 and 48 h after the end of the exposure period. No allergic skin reactions were observed at 24 and 48 h after the end of the challenge procedure; the sensitisation rate was 0 %. It can be assumed that the same applies to tributyl citrate (CAS 77-94-1) as it is a near analogue to the test substance acetyltri-2 -ethylhexylcitrate (ATEHC).

In the "toxicity profile of triethyl citrate" (TNO BIBRA, 1998) it is stated that triethyl citrate is not a skin sensitizer in guinea pigs (Fassett, undated; Ohtaki et al., 1985; Sheftel, 1990). No further details are available. In the same document it is also stated that in an unsuccessful attempt to induce sensitisation in a maximisation test (Kligman, 1966; Kligman & Epstein, 1975), no sensitisation reactions were produced in 22 volunteers treated with 20 % triethyl citrate in petrolatum (Epstein, 1975). The maximisation test involves an induction phase of five consecutive 48 -hr covered patch tests, sometimes separated by 24 -hr periods of treatment with a mild irritant, followed 10 - 14 days later by a final 48 -hr closed patch test with the same concentration. No further details are available. In the document "Opinion on the toxicological characteristics and risks of certain citrates and adipates used as a substitute for phthalates as plasticisers in certain soft PVC products" [Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE), 1999] the following is stated: The substance appears to be a strong sensitizer in the guinea pig maximisation test. Further in the same document the following is stated: It (triethyl citrate) did not show any evidence of sensitising capacity or skin irritation in humans, but there are no further details available. It can be assumed that the same applies to tributyl citrate (CAS 77-94-1) as it is a near analogue to the test substance triethyl citrate.

Taking all these result into account, it can be concluded that tributyl citrate is not sensitising to skin.


Migrated from Short description of key information:
The following results are available from related substances:
1) ATEHC was not sensitising to rabbits in a GLP test according to OECD 406 (Guinea pig maximisation test). [Read-across data from acetyltri-2-ethylhexylcitrate (CAS 144-15-0)]
2) & 3) BIBRA (1998): Triethyl citrate was not sensitising in guinea pigs and humans. [Read-across data from triethyl citrate (CAS 77-93-0)]
4) & 5) CSTEE (1999): Triethyl citrate was sensitising in guinea pigs, but not in humans. [Read-across data from triethyl citrate (CAS 77-93-0)]

Justification for selection of skin sensitisation endpoint:
GLP and guideline study with the structural analogue ATEHC.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no indication that TBC may cause respiratory sensitisation.


Justification for selection of respiratory sensitisation endpoint:
No further study is needed since TBC is not a sensitiser and it is marginally available in the air for inhalation.

Justification for classification or non-classification

Skin sensitisation

The test material does not meet the criteria for classification and will not require labelling as skin sensitising in accordance with European Regulation (EC) No. 1272/2008.