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Description of key information

An OECD 406 and OECD 429 study was conducted on 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters and determined not to be a skin sensitiser in either study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25 June 2013 to 10 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with EU and OECD Guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon., UK
- Age at study initiation: Eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd, Oxon., UK
- Water: Free access to mains tap water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve limits of 17 to 23 degrees C.
- Humidity (%): The humidity was set to achieve 30 to 70%.
- Air changes (per hr): At least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to 12 hours continous light (06:00 to 18:00) and 12 hours darkness.

IN-LIFE DATES: From: 25 June 2013 To: 10 July 2013
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Concentrations: Undiluted test item, 50% v/v in acetone/olive oil (4:1), 25% v/v in acetone/olive oil (4:1).
No. of animals per dose:
4 per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: No data.
- Irritation: No signs of visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thiickness were noted.No systemic toxicity occurred.
- Lymph node proliferation response: No data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4: 1. This vehicle was chosen as it produced a suitable formulation at the required concentration.

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
25% in in acetone/olive oil 4:1, Stimulation Index: 7.33, Result: positive.
a-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Parameter:
SI
Remarks on result:
other: 25 % v/v in acetone/olive oil 4:1: Stimulation Index: 0.97, Result: Negative 50 % v/v in acetone/olive oil 4:1: Stimulation Index: 1.27, Result: Negative 100 % v/v in acetone/olive oil 4:1: Stimulation Index: 1.58, Result: Negative
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table below.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index:

Concentration

(% v/v) in acetone/olive oil 4:1

 

dpm

 

dpm/Nodea

  Stimulation Indexb

 

Result

 

Vehicle

 

6682.19

 

835.27

 

na

 

na

 

25

 

6504.92

 

813.12

 

0.97

 

Negative

 

50

 

8467.35

 

1058.42

 

1.27

 

Negative

 

100

 

10530.45

 

1316.31

 

1.58

 

Negative

dpm = Disintegrations perminute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals was treated with acetone/olive oil 4: 1 alone.

 

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

25 % v/v in acetone/olive oil 4:1: Stimulation Index: 0.97, Result: Negative

50 % v/v in acetone/olive oil 4:1: Stimulation Index: 1.27, Result: Negative

100 % v/v in acetone/olive oil 4:1: Stimulation Index: 1.58, Result: Negative

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1989-11-28 to 1989-12-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test was performed before REACH was inforce.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: not mentioned
- Weight at study initiation: 380 g (mean weight of the test animals); 376 g (mean weight of the control animals)
- Housing: 1 - 5 animals in Makrolon cages type IV
- Diet (e.g. ad libitum): G4 complete feed for guinea pigs ad libitum, supplied by Ssniff Spezialfutter GmbH, Soest, Germany
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 5 - 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 1
- Humidity (%): 60 ± 5
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route:
intradermal
Vehicle:
maize oil
Concentration / amount:
10 %
Day(s)/duration:
7
Adequacy of induction:
not specified
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
2
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#4
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#5
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#6
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#7
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#8
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#9
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#10
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#11
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#12
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#13
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#14
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#15
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#16
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#17
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#18
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#19
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No.:
#20
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
test group: 20 animals
control group: 10 animals
Details on study design:
RANGE FINDING TESTS:
A test substance concentration of 100% were tested in a preliminary study, no further details were mentioned

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: 1st exposure: intracutaneous injections (0.1 cm³); 2nd exposure: epicutaneous for 48 hours; evaluation after 1 and 24 hours each
- Concentration in Freunds Complete Adjuvants (FCA): 10% test substance in a mixture of FCA and maize germ oil (1:1)
- Test group: dermal: undiluted test substance (2 x 4 cm patch)
- Control group: maize germ oil (2 x 4 cm patch)
- Frequency of applications: day 0: intradermal treatment, day 6: pretreatment with sodium dodecyl sulphate (10% in vaseline) and day 7: dermal treatment
- Duration: 3 weeks
- Concentrations: 100% test substance

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: on day 21
- Exposure period: dermal application for 24 hours
- Test groups: undiluted test substance (2 x 2 cm patch)
- Control group: undiluted test substance (2 x 2 cm patch)
- Site: left flanks
- Concentrations: 100% v/v
- Evaluation (hr after challenge): 48 and 72 hours after application
Challenge controls:
see above B. Challenge exposure
Positive control substance(s):
not specified
Positive control results:
not carried out
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Group:
positive control
Remarks on result:
not measured/tested

RESULTS OF PILOT STUDY: no irritation at concentrations of 100% test substance

RESULTS OF TEST

- Sensitization reaction: 0/20

- Clinical signs: Local reactions after intradermal application (test and control animals after 1 and 24 hours): All FCA treated injections sites showed severe erythema, edema and necrosis. The animals treated with 10% test substance in maize germ oil showed well defined erythema and edema.

Local reactions after patch test (48 hours): 1 hour after patch removal, test and control animals: whole application area showed erythema and edema, as well as inflamed or bloody lesions, restlessness of the animals and scratching in the application area.

24 hours after patch removal: Some animals showed erythema and eschar formation in the whole application area.

Interpretation of results:
GHS criteria not met
Conclusions:
1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters showed no sensitising effect on guinea pig skin under the described test conditions.
Executive summary:

1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was tested for dermal sensitisation in guinea pigs by the Magnusson and Kligman maximisation test according to OECD guideline 406 (12 May 1981).

The sensitising potential of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was determined using a test group of 20 and a control group of 10 animals. All reactions, especially the formation of erythema and oedema, were assessed 48 and 72 hours after the challenge treatment. The maximum concentration which caused no dermal irritation in a prelinminary test was found to be 100% test substance (as supplied).

The following concentrations of test substance were used for the induction treatments: the test substance was employed in a 10% mixture with maize germ oil for the intracutaneous injection, while the 100% test substance (as supplied) was used for the dermal treatment.

The preliminary test showed that the undiluted test substance caused no dermal irritation. In order to induce slight to moderate dermal inflammation, 24 hours before the dermal induction treatment the skin in the injection area was pretreated with sodium dodecyl sulphate (10% in vaseline).

All FCA treated injections sites (diluted 1 +1 with maize germ oil), assessed 1 and 24 hours after intradermal induction treatment, showed severe erythema, edema and necrosis. The control animals had comparable signs of irritation at the injection sites.

The reactions to the dermal induction treatment for 48 hours were described as follows: 1 hour after patch removal, test and control animals showed erythema and edema in the whole application area, as well as inflamed or bloody lesions, the animals were restless and showed scratching in the application area. 24 hours after patch removal some animals showed erythema and eschar formation in the whole application area.

The first challenge treatment was carried out with the undiluted test substance (100%). No signs of dermal irritation were found on the test animals or the control animals 48 and 72 hours after the challenge treatment. No second challenge was carried out.

On the basis of these results, 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was assessed as having no sensitising effect on the skin of guinea pigs.

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

In the key study according to OECD 406 to assess the sensitising properties of 1,2,4 -benzenetricarboxylic acid, decyl octyl ester a maximisation test in guinea pigs has been conducted with test concentrations of 10% (induction) and 100% (challenge). No evidence of skin sensitisation was observed in any of the treated animals.

In a supporting study a LLNA-test according to OECD 429 was conducted with the test item.

1,2,4 -benzenetricarboxylic acid, decyl octyl ester was considered to be a non-sensitizer under the conditions of the test.

Justification for classification or non-classification

The results of the sensitisation tests show that1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triestersdoes not need to be classified according to

EU regulations (Directive 67/548/EEC and Regulation (EC) No 1272/2008.

There are data lacking for the classification of 1,2,4 -Benzenetricarboxylic acid, decyl octal ester with regard to respiratory

sensitisation.