Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid

Endpoint summary

Description of key information

In a test according to OECD 406 (Buehler) 20 female guinea pigs were exposed to the undiluted Bis(2-ethylhexyl) azelate on day 0, 6 and 13 during 6 hours epicutaneously under occlusion (Phycher 2017). A group of 10 female guinea pigs received a similar treatment with liquid paraffin on day 0, 6 and 13. Both groups were challenged with undiluted substance on day 26 during 6 hours epicutaneously under occlusion and were checked 24, 48 and 72 hours thereafter for signs of skin reaction.

In the treated group (treatment dose of 100%), no macroscopic cutaneous reactions attributable to allergy were observed during the examination following the removal of the occlusive dressing.

In the control group (associated with the treatment dose of 100%), no cutaneous intolerance reactions were observed during the examination following the removal of the occlusive dressing.

The skin sensitising potential of Bis(2-ethylhexyl) azelate (CAS 103-24-2) was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD guideline 429 and in compliance with GLP (Bradshaw, 2012).

Based on a range-finding test in one female CBA/CaOlaHsd mouse, the undiluted test substance (100%) and concentrations of 25% and 50% (v/v) in acetone/olive oil (4:1 v/v) were selected for the treatment of mice in the main study. In this experiment, 4 female CBA/CaOlaHsd mice per test group were treated with the test substance or vehicle alone, respectively. The test substance or the vehicle were applied on the external surface of each ear (25 µL/ear) for three consecutive days. Five days after the first topical application, the cell proliferation of pooled lymph nodes per group was measured by incorporation of ³H-methyl thymidine and expressed as the amount of radioactive disintegration per minute (DPM). The DPM/lymph node for each test group were 5244.83, 5178.42 and 8048.58 at concentrations of 25%, 50% and 100% of the test substance, respectively. For the control group, a DPM/lymph node of 1553.82 was determined. Based on these results, stimulation indices of 3.38, 3.33 and 5.18 were calculated for treatment concentrations of 25%, 50% and 100%, respectively. No local or systemic toxicity and no effects on body weights were observed.

The reactivity of the substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm (BASF 2014).

The mean C-peptide depletion, caused by the test substance was determined to be 0.52%.

The mean K-peptide depletion, caused by the test substance was determined to be -1.05%.

The substance is considered non-sensitizing under the conditions of the test.

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:

20 February 2017 to 30 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The results of in vitro assays are indicative for no sensitization, but an LLNA test showed ambiguous results. Therefore in order to exclude false positive results in the LLNA test this additional in vivo test was performed.
The standard study plan related to this study was approved by the registered Ethics Committee No. 76.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ENVIGO (Kreuzelweg 53, 5961 NM HORST The Netherlands
- Age at study initiation: 3-4 weeks
- Weight at study initiation: 270-306 g
- Housing: groups of maximum 3 in polycarbonate cages.
- Diet: ENVIGO, 2040C ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ° C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route:

epicutaneous, occlusive

Vehicle:

other: liquid paraffin

Remarks:

50% gave colorless solution

Concentration / amount:

100%

Day(s)/duration:

6

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: liquid paraffin

Concentration / amount:

100%

Day(s)/duration:

6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 treatment and 10 controls

Details on study design:

RANGE FINDING TESTS:
Three guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 6 hours at 4 different concentrations: 100%, and diluted at 75%, 50% and 25% in liquid paraffin.
Washing of the skin after removal of the dressing was done with liquid paraffin.
maximal non-irritant concentration was 100%

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours (on day 0, 6 and 13)
- Test groups: 20 females treated with 0.5 mL 100% undiluted substance
- Control group: 10 females treated with 0.5 mL liquid paraffin
- Site: scapular zone
- Frequency of applications: 3 times

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: on day 26
- Exposure period: 6 hours
- Test groups: 20 females treated with 0.5 mL 100% undiluted substance
- Control group: 10 females treated with 0.5 mL 100% undiluted substance
- Site: dorso-lumbar zone of the left flank
- Evaluation (hr after challenge): at 24, 48 and 72 hour

Challenge controls:

0.5 mL of liquid paraffin was applied on the right flank.

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde in 3 separate tests

Positive control results:

positive in 3 applications

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 (100% in challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% (100% in challenge)

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:

48

Group:

negative control

Dose level:

0 (challenge 100%)

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:

48

Group:

test chemical

Dose level:

100% (100% in challenge)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0 (100% challenge)

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:

72

Group:

test chemical

Dose level:

100% (100% in challenge)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

On the challenge control sites (challenged at the other flank with liquid paraffin) one animal was observed with discrete to intense erythema at the 24 and 48 hours reading. This effect was fully reversible at the 72 hours reading.

groups	Time (h)	Concentrations	Incidence				% of positive responses	% of sensitized
			0	1	2	3
Control Group 1	24	100%	10	0	0	0	0%
	48	100%	10	0	0	0	0%
	72	100%	10	0	0	0	0%
	24	0%	10	0	0	0	0%
	48	0%	10	0	0	0	0%
	72	0%	10	0	0	0	0%
Treated Group 2	24	100%	20	0	0	0	0%	0%
	48	100%	20	0	0	0	0%	0%
	72	100%	20	0	0	0	0%	0%
	24	0%	19	0	0	1	5%	0%
	48	0%	19	1	0	0	5%	0%
	72	0%	20	0	0	0	0%	0%

 

Grading scale

0....... No visible change

1....... Discrete or patchy erythema

2....... Moderate and confluent erythema

3....... Intense erythema and swelling

Interpretation of results:

GHS criteria not met

Conclusions:

In a Buehler test the substance was not sensitizing to the skin.

Executive summary:

In a test according to OECD 406 (Buehler) 20 female guinea pigs were exposed to the undiluted substance on day 0, 6 and 13 during 6 hours epicutaneously under occlusion. A group of 10 female guinea pigs received a similar treatment with liquid paraffin on day 0, 6 and 13. Both groups were challenged with undiluted substance on day 26 during 6 hours epicutaneously under occlusion and were checked 24, 48 and 72 hours thereafter for signs of skin reaction.

In the treated group (treatment and challenge dose of 100%), no macroscopic cutaneous reactions attributable to allergy were observed during the examination following the removal of the occlusive dressing.

In the control group (associated with the challenge dose of 100%), no cutaneous intolerance reactions were observed during the examination following the removal of the occlusive dressing.

No cutaneous reaction was recorded in animals from the control group after the challenge phase, on the treated area with liquid paraffin.

In conclusion, the substance does not have to be classified in category 1 as a skin sensitiser, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The analogue Bis(2-ethylhexyl) azelate is not sensitizing to the skin (weight of evidence based on an ambiguous LLNA test and negative in vitro and Buehler tests). Uptake via the skin for this source substance is expected to be limited and both azelaic acid and ethylhexanol are not sensitizing. As the other hydrolysis products of Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid also do show sensitizing properties and the toxicokinetic behaviour of the substance is very similar to that of Bis(2-ethylhexyl) azelate, no sensitization is expected for Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the information available, no classification for sensitization according to Regulation EC No 1272/2008 (CLP) for Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid is considered necessary.