Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Endpoint summary

Description of key information

Skin sensitisation: sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 26 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: mains tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

Preliminary screening test: 100%
Main test: 25, 50 and 100% (v/v)

No. of animals per dose:

Preliminary screening test: 1
Main test: 4

Details on study design:

RANGE FINDING TESTS: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the Draize scoring system. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post-dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
- Compound solubility: The test item was soluble in in acetone/olive oil (4:1 v/v) at 50% and the solution considered suitable for dosing.
- Clinical signs and Irritation: No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on the results of the preliminary screening test, the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-Methylthymidine incorporation determined by β-scintillation.
- Criteria used to consider a positive response: 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).
A test item was regarded as a sensitiser if at least one concentration of the test item resulted 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 was classified as a "non-sensitiser".
The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3-fold increase in ³HTdR incorporation. The equation used for the calculation of EC3 is:
EC3= c + [[(3−d)/(b−d)] x (a−c)]
where:
a = lowest concentration giving stimulation index > 3
b = actual stimulation index caused by ‘a’
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by ‘c’

TREATMENT PREPARATION AND ADMINISTRATION:
Animals were treated with the undiluted test item, the test item at concentrations of 25% and 50% (v/v) in acetone/olive oil (4:1 v/v) or the vehicle alone by daily application of 25 µL of the appropriate solution to the dorsal surface of each ear for three consecutive days (Days 1-3). Five days following the first topical application of the test item or vehicle (Day 6), each animal was injected with 250 µL of phosphate buffered saline (PBS) containing 20 µCi ³H-TdR (concentration: 80 µCi/mL, specific activity: 2.0 Ci/mmol) via the tail vein. Five hours following administration of ³H-TdR, animals were sacrificed and the draining auricular lymph nodes were excised and pooled for each experimental group (total: 8 nodes/group). A single cell suspension of pooled lymph node cells was prepared in PBS by gentle mechanical disaggregation through 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish and each lymph node cell suspension was transferred into a centrifuge tube. The petri dish was washed with PBS to remove all remaining lymph node cells, and pooled lymph node cells were pelleted by centrifugation. The pellet was resuspended in PBS and re-pelleted. To precipitate macromolecules incorporating radioactive material, the pellet was resuspended in 5% trichloroacetic acid (TCA). After approx. 18 h incubation at 4°C, the precipitates were recovered by centrifugation, resuspended in TCA and transferred to scintillation fluid. ³H-TdR incorporation was measured by β-scintillation counting.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

Treatment with the current positive control substance α-Hexylcinnamaldehyde, technical product (85%) at 25% in acetone:olive oil (4:1 v/v) resulted in a stimulation index (SI) of 5.76, thus meeting the reliability criteria for the LLNA (SI > 3).

Key result

Parameter:

SI

Value:

2.21

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

2.16

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

4.27

Test group / Remarks:

100%

- Clinical Observations and Mortality Data

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

- Bodyweight

Body weight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period except for one animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1 which showed a greater than expected body weight loss (-13% of initial body weight).

- Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1.

 

Table1. Disintegrations per Minute (dpm), Disintegrationsper Minute/Node and Stimulation Index

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/Node*	Stimulation Index	Result
Vehicle	10638.52	1329.82	na	na
25	23466.12	2933.27	2.21	Negative
50	22995.27	2874.41	2.16	Negative
100	45373.88	5671.74	4.27	Positive

 

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

na: not applicable

 

- Calculation of EC3 value

EC3= c + [[(3−d)/(b−d)] x (a−c)]

a = 100

b = 4.27

c = 50

d = 2.16

EC3= 50 + [[(3−2.16)/(4.27−2.16)] x (100−50)] = 70

The concentration of test item expected to cause a 3-fold increase in ³HTdR incorporation (EC3 value) was calculated to be 70%.

Interpretation of results:

other: Skin Sens. 1B, H317. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 - 30 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted 04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 and 258 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier of synthetic peptides: GenScript, Picataway, NJ, USA and RS Synthesis, Louisville, KY, USA
- Preparation of peptide stock solutions:
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

VEHICLE CONTROL
- Substance: acetonitrile
- Justification for selecting vehicle: the test substance was soluble in the vehicle

POSITIVE CONTROL
- Substance: ethylene glycol dimethacrylate
- Preparation: the positive control was prepared as 50 mM solution in acetonitrile.

STABILITY CONTROL
Stability control of both peptides was prepared at a concentration of about 0.5 mM.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature used during treatment / exposure: room temperature
- Duration of treatment / exposure: at least 24 ± 2 h in the dark

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Liquid chromatograph Agilent HP 1100 with DAD
- Column: Phenomenex Luna 3μ C18 (2), 100 mm x 2 mm with guard column „Security Guard“ C18, 4 mm x 2 mm
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water, HPLC grade
B: 0.085% (v/v) trifluoracetic acid in acetonitrile, HPLC grade
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 25
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258nm
- Injection volume: 2 µL
- Detection: Diode Array Detector
- Peptide standards: calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test substance incubation, were measured in parallel with the test substance samples

Run / experiment:

other: Experiment 1

Parameter:

other: % depletion of cysteine-containing peptide (mean value of 3 replicates)

Value:

-0.33

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

59.06% depletion of cysteine-containing peptide (mean value of 3 replicates)

Remarks on result:

other: the test substance showed minimal peptide reactivity for the cystein-containing peptide

Run / experiment:

other: Experiment 1

Parameter:

other: % depletion of lysine-containing peptide (mean value of 3 replicates)

Value:

-2.75

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

16.77% depletion of lysine-containing peptide (mean value of 3 replicates)

Remarks on result:

other: the test substance showed minimal peptide reactivity for the lysine-containing peptide

Other effects / acceptance of results:

OTHER EFFECTS:
The test substance was solved in acetonitrile. The samples of the test substance with the peptides were emulsions. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the C-peptide.
No co-elution of the test substance and peptides occurred as demonstrated by the consistent values of the area ratios 220 nm/258 nm

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, depletion of cysteine and lysine-containing peptide (mean value of 3 replicates) was within the range of the historical control data
- Acceptance criteria met for positive control: yes, depletion of cysteine and lysine-containing peptide (mean value of 3 replicates) was within the range of the historical control data

The negative and positive control fall within the range of the historic control data.

Table 1: Historic Range of Negative Control (Jan 2012 - Aug 2014, n = 61)

Acetonitril	mean peptide concentration [mM]	SD
Cysteine-peptide	0.485	0.025
Lysine-peptide	0.501	0.016

Table 2: Historic Range of Positive Control (Feb 2012 - Aug 2014, n = 50)

Ethylene glycol dimethacrylate (50 mM)	mean peptide concentration [mM]	SD	mean peptide depletion [%]	SD
Cysteine-peptide	0.197	0.050	59	10
Lysine-peptide	0.433	0.029	14	5

Table 3: Reaction with cysteine-peptide

	peak area [mAU*s] at 220 nm			peptide concentration [mM]					peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	mean	SD	Sample 1	Sample 2	Sample 3	mean	SD
Negative Control	598.6	586.6	574.7	0.475	0.465	0.456	0.465	0.009	- 2.03	0.01	2.02	0.00	2.03
Test substance	596.5	586.3	582.9	0.473	0.465	0.463	0.467	0.006	- 1.67	0.05	0.62	- 0.33	1.19
Positive Control	270.1	239.9	206.1	0.215	0.192	0.165	0.191	0.025	53.72	58.86	64.60	59.06	5.44

Table 4: Reaction with lysine-peptide

	peak area [mAU*s] at 220 nm			peptide concentration [mM]					peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	mean	SD	Sample 1	Sample 2	Sample 3	mean	SD
Negative Control	685.3	664.5	700.5	0.503	0.488	0.515	0.502	0.013	- 0.28	2.78	- 2.50	0.00	2.65
Test substance	700.8	698.1	707.5	0.515	0.513	0.520	0.516	0.004	- 2.56	- 2.16	- 3.54	- 2.75	0.71
Positive Control	576.7	569.5	561.8	0.423	0.418	0.412	0.418	0.006	15.68	16.74	17.87	16.77	1.10

Interpretation of results:

other: DPRA prediction: negative (minimal reactivity) according to OECD TG 442C

Conclusions:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed minimal peptide reactivity.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 Nov - 21 Dec 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of The United Kingdom, UK

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same Regulation, the data was included to avoid unnecessary testing.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Staffordshire, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 329 - 396 g (range)
- Housing: the animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet: Guinea Pig FD1 Diet (Special Diets Services Limited, Essex, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 46 - 67
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

10%

Day(s)/duration:

Single injection on Day 1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

Applied on Day 7, 48 h

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

75% (v/v) and undiluted

Day(s)/duration:

Applied on Day 21, 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 (test group)
5 (controls)

Details on study design:

RANGE FINDING TESTS: To determine a suitable concentration for the intradermal induction in the main study, 4 concentrations of the test substance (1, 5, 10 and 25%) in arachis oil BP were selected. A total of 4 female guinea pigs each received 4 injections of 0.1 mL of one concentration only. A concentration of 10% in arachis oil BP induced erythema (grade 1) after seven days, therefore this concentration was selected for the intradermal induction stage of the main study. To determine a suitable concentration for the epicutaneous induction in the main study, 2 females were intradermal injected with Freund's Complete Adjuvant on Day 0, and then each treated by epicutaneous application with 25, 50 and 75% v/v of the test substance in arachis oil BP, and the undiluted test substance. The test substance was applied to the clipped flanks of the animals under occlusive dressings for 48 hours, and the degree of erythema and oedema was assessed approximately 1, 24 and 48 h after patch removal. The undiluted test material was selected for the main study topical induction, as no erythema or oedema were observed after 48 h. To determine a suitable concentration for the epicutaneous challenge in the main study, the undiluted test material and 3 concentrations (25, 50 and 75%) in arachis oil BP were selected. The test substance was applied to the clipped flanks of two females under occlusive dressings for 24 h and the degree of erythema and oedema was assessed approximately 1, 24 and 48 h after patch removal. The undiluted test material and a concentration of 75% in arachis oil BP were selected for the main study topical induction, as no erythema or oedema was observed after 48 h.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: a 10% (w/v) formulation of the test substance in arachis oil BP
Injection 3: a 10% (w/v) formulation of the test material in a 1:1 mixture (v/v) FCA/water
Epicutaneous: the undiluted test substance
- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: arachis oil BP
Injection 3: a 50% (w/v) formulation of arachis oil BP in a 1:1 mixture (v/v) FCA/water
Epicutaneous: blank patch
- Site: shoulder region (intradermal and epicutaneous)
- Frequency of applications: Day 0 (intradermal) and Day 7 (epicutaneous)
- Duration: Days 0 - 7
- Concentrations: intradermal 10%, epicutaneous undiluted test substance

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day(s) of challenge: 21
- Exposure period: 24 h
- Test groups: test substance in arachis oil BP and test substance only
- Site: right flank (test substance only), left flank (test substance in vehicle)
- Concentrations: 75% and undiluted
- Evaluation: 24 and 48 h after patch removal

OTHER: Approximately 24 and 48 h after intradermal injection the degree of erythema at the injection sites was evaluated. The degree of erythema and oedema was assessed approximately 1 and 24 h after patch removal following the topical induction. Any evidence of systemic toxicity was recorded. The bodyweight of each animal was recorded at the start and end of the study.

Positive control substance(s):

yes

Remarks:

reliability checks were performed at least every 6 months, with a suitable substance

Positive control results:

Reliability checks were performed at least every 6 months at the testing laboratory to show the sensitivity and reliability of the study results. A summary of the studies performed from July 1995 to June 1997 under same experimental conditions was included in the study report. The animals were administered 2,4-dinitrochlorobenzene, neomycin sulphate or 2-mercaptobenzothiazole. A response of 60-100% of the animals was observed in all the reliability checks. Therefore the positive control is considered to be valid.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induction: 10% (intradermal), undiluted (epicutaneous); challenge: 75% and undiluted

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No local skin irritation reactions and no signs of systemic toxicity were observed

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induction: 10% (intradermal), undiluted (epicutaneous); challenge: 75% and undiluted

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No local skin irritation reactions and no signs of systemic toxicity were observed

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Induction: 0%; challenge: 75% and undiluted

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No local skin irritation reactions and no signs of systemic toxicity were observed

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Induction: 0%; challenge: 75% and undiluted

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No local skin irritation reactions and no signs of systemic toxicity were observed

The administration of the 75 % (w/v) solution of the test item in vehicle to the left flank and the undiluted test substance to the right flank did not cause any skin irritation signs in the animals of the treatment group or the control group following the challenge treatment. No systemic toxicity was observed during the course of investigation and the mean value for body weight was comparable between the treatment group and control group.

Very slight erythema (grade 1) was noted at the intradermal induction sites in 10/10 animals after 24-hour and in 7/10 animals after 48-hour observation. 3/5 animals after 24 h and 1/5 animals after 48 h, respectively, of the control group showed very slight erythema (grade 1) at the intradermal induction at the 24-hour and 48-hour observation.

Well-defined to moderate to severe erythema (grade 2) with slight oedema (grade 2) was noted at the topical induction sites in 10/10 animals and bleeding from the intradermal injection sites was noted in 3/10 animals after 1 h observation. Very slight erythema (grade 1) with or without very slight oedema (grade 0 or 1) was noted at the topical induction sites in 8/10 animals and small superficial scattered scabs were noted in 2/10 animals after 24-hour observation.

No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo data

The skin sensitisation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate was assessed in a Local Lymph Node Assay (LLNA) performed according to OECD guideline 429 and in compliance with GLP (Harlan, 2012c). Four female mice per group were treated with 50 µL (25 µL per ear) of the vehicle (acetone/olive oil 4:1) of the test material at 25%, 50% and in undiluted form for 3 consecutive days. Five days after the first application, animals were injected with ³H-methylthymidine (³HTdR) at 20 µCi/mouse. The draining auricular lymph nodes from all mice were excised 5 h after injection and pooled for each treatment group. Single cell suspensions of pooled lymph node cells were prepared for determination of ³HTdR incorporation byβ-scintillation counting. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/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, SI). No mortality occurred and no clinical signs of systemic toxicity were observed in any animal during the study period. No changes in body weight were noted except for one animal treated with the test material at 50%, which showed a greater than expected body weight loss (-13% of initial body weight). Treatment with vehicle and test material at 25, 50 and 100% resulted in DPM/node values of 1329.82, 2933.27, 2874.41 and 5671.74, respectively. The corresponding SI values for the low-, mid- and high-dose groups were 2.21, 2.16 and 4.27, respectively. The concentration of test item expected to cause a 3-fold increase in ³HTdR incorporation (EC3 value) was calculated to be 70%. The reported historical positive control data, including the most recent test withα-hexylcinnamaldehyde, tech., 85%, fulfilled the reliability criteria for the LLNA (SI > 3). The test material was therefore considered to be sensitising under the conditions of the test.

 

A Guinea pig maximisation test was performed with the test substance according to OECD guideline 406 and under GLP conditions (Safepharm, 1998). A range-finding study was performed to establish suitable dose levels for the induction and challenge treatments. In the main study, 10 female Dunkin-Hartley guinea pigs were induced intradermally on Day 0 with 10% test substance in arachis oil BP on both sides of the spine with and without Freund’s Complete Adjuvant. On day 7, the undiluted test substance was applied to the skin of the animals for 48 hours under occlusive conditions. On Day 21, the challenge treatment was performed by topical application of the test substance in undiluted form and as a 75% dilution for 24 hours under occlusive conditions. Five animals served as negative controls and were exposed to the vehicle only according to the same protocol as the treatment animals. Reliability checks were performed with 2,4-dinitrochlorobenzene, neomycin sulphate or 2-mercaptobenzothiazole under the same experimental conditions and indicated the reliability and sensitivity of the study. Following the intradermal induction, very slight erythema was observed at the injection sites of 10/10 treatment animals (10% test material in arachis oil) and 3/5 control animals (arachis oil) at the 24-hour reading time point, and 7/10 treatment animals and 1/5 control animal at the 48-hour reading time point. Following the topical induction, well-defined to severe erythema and slight oedema was observed at the application site in 10/10 treatment animals at the one-hour reading time point; and very slight erythema was noted in 8/10 treatment animals while 4/10 showed very slight oedema at the 24-hour reading time point. Bleeding from the intradermal induction site was noted in 3/10 treatment animals at the one-hour reading time point following the topical induction, while scabs were noted at the topical induction sites in 2/10 treatment animals at the 24-hour observation time point. No control animals showed erythema and oedema following the topical induction, however, bleeding from the intradermal induction site was noted in 1/5 control animals at the one-hour reading time point. At the challenge, 0/10 treatment animals and 0/5 control animals showed skin reactions at the challenge site 24 and 48 h after the challenge treatment, indicating that the test substance does not cause skin sensitisation. Under the conditions of this test the test substance is considered to be not sensitising to guinea pigs.

In chemico data

The protein reactivity of the test substance was assessed in a Direct Peptide Reactivity Assay, performed according to OECD 442C and in compliance with GLP (BASF SE, 2014). 100 mM of test substance in acetonitrile was incubated with lysine-and cysteine -containing standards for 24 h at room temperature in the dark. The relative peptide concentration was measured by HPLC with gradient elution and UV detection by photodiode array detector at 220 and 258 nm. The value for cysteine-containing (-0.33%) and lysine-containing (-2.75%) peptide depletion was within the range of the negative control (0.00%). The incubation with the positive control ethylene glycol dimethacrylate caused a strong increase of the cysteine-containing (59.06%) and lysine-containing (16.77%) peptide depletion compared to the negative control (0.00%). Under the conditions of the Direct Peptide Reactivity Assay the test substance showed minimal peptide reactivity.

Conclusions for skin sensitisation

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate showed minimal peptide reactivity in the Direct Peptide Reactivity Assay. Additional in vitro testing was performed with the ARE reporter (LuSens) assay (similar to OECD guideline 442D), which showed a significant induction of luciferace activity, and the h-CLAT assay (OECD guideline 442E), which showed no induction of dendritic cell activation. As the test substance falls outside the applicability range for the ARE reporter assay and the h-CLAT assay, due to the log Pow value, the results have been disregarded and are not reported. In the GPMT, the test substance did not show skin sensitising properties. The result of the LLNA was positive, with an EC3 value of 70%. In a worst-case approach the result of the LLNA is used for the hazard assessment and propane-1,2,3-triyl 3,5,5-trimethylhexanoate is considered to have skin sensitising potential.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on the skin sensitisation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate meet the classification criteria for Skin sensitisation Category 1B (H317: May cause an allergic skin reaction) according to Regulation (EC) No. 1272/2008. There are no data available on respiratory sensitisation.