2-ethylhexyl hydrogen maleate

Endpoint summary

Description of key information

Based on the fïndings in the adjuvant sensitisation test in guinea pigs according to OECD guideline 406, the test substance is judged to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-10-12 to 2006-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

30 July 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Ltd, 4452 Itingen, Switzerland

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was conducted due to non-REACH regulatory requirements. With the existing data from this valid Maximization test according to OECD Guideline 406 not only being acceptable but of good quality (Klimisch Score 1), this study precludes the need for an additional LLNA study. In addition, a supplementary LLNA study would violate the ECHA objectives with regards to animal welfare.

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Albino Dunkin Hartly guinea Pig, CRL:(HA)BR, SPF

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kisslegg, Germany
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 340 - 388 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Provimi Kliba 3418, batch nos. 43/05 and 62/05, guinea pig breeding / maintenance diet, containing Vitamin C (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum.
- Water: Community tap water from Füllinsdorf, ad libitum
- Acclimation period: 12 October 2005 to 25 October 2005; Under laboratory conditions after health examination. No acclimation for the animals of the pretest.
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Concentration / amount:

Test item at 5 %

Day(s)/duration:

Day 1

Adequacy of induction:

other: highest technically applicable concentration causing moderate skin irritation.

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Concentration / amount:

Test item at 15 %

Day(s)/duration:

Day 8/ 48 h

Adequacy of induction:

other: highest concentration causing mild skin irritation.

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Concentration / amount:

Test item at 10%

Day(s)/duration:

Day 22/ 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

5 animals in control group
10 animals in test group

Details on study design:

RANGE FINDING TESTS:
Intradermal Induction:
The 5 % concentration of test item used for the intradermal induction exposure was well-tolerated systemically and was the highest technically applicable concentration causing moderate skin irritation.

Epidermal Induction:
The 15 % concentration of test item used for the epidermal induction exposure was well-tolerated systemically and was the highest to cause mild skin irritation. Higher concentrations such as 100, 75, 50 and 25 % tested during the epidermal pretest I showed mild to intense erythema with/without crusts and oedema.

Epidermal Challenge:
The 10 % concentration of the test item used for the challenge application was the maximum tested non-irritant concentration. To determine the different concentrations one intradermal and two epidermal pretests were performed.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (one intradermal and one epidermal)

1) INTRADERMAL INJECTIONS
Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
- Test groups:
a) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
b) The test item at 5 % in PEG 300.
c) The test item at 5 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Control group:
a) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
b) PEG 300
c) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Site: An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. The intradermal injections were made at the border of a 4 x 6 cm area in the clipped region
- Concentrations: Test item at 5% in PEG 300

2) EPIDERMAL APPLICATIONS
- Exposure period: The occlusive dressings were left in place for 48 hours.
- Test groups: A 2 x 4 cm patch of filter paper was saturated with the test item at 15 % in PEG 300 and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape.
- Control group: The guinea pigs of the control group were treated as described above with PEG 300 only, also applied at a volume of approximately 0.3 mL.
- Site: The scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was used.
- Duration: The reaction sites were assessed 24 and 48 hours after removal of the bandage for ery-thema and oedema according to the method of Magnusson and Kligman.
- Concentrations: Test item at 15% in PEG 300.

B. CHALLENGE EXPOSURE (Day 22)
- No. of exposures: 1
- Day(s) of challenge: Day 22 (two weeks after epidermal induction)
- Exposure period: 24 h
- Test groups: Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 10 % (applied to the left flank) using the same method as for the epidermal application. The volume of test item preparation was approximately 0.2 mL.
- Control group: Two patches (3 x 3 cm) of filter paper were saturated with the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of vehicle applied was approximately 0.2 mL.
- Site: Left flank (test substance, 10%) and right flank (vehicle only) of each guinea pig.
- Concentrations: 10 % (highest tested non-irritating concentration)
- Evaluation (hr after challenge): Three hours later (approximately 48 hours from the start of the challenge application) the skin reaction was observed and recorded according to the numerical grading system described in the section "Any other information on results incl. tables". Approximately 24 hours after this observation a second observation (approximately 72 hours from the start of the challenge application) was made and once again recorded.

Challenge controls:

yes

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde

Positive control results:

Based on the results of an adhuvant sensitisation test in guinea pigs and in accordance with Commisiion 2001/59/EC, alpha-Heylcinnamaldehyde at 1% does have to be classified and labelled as skin sensitizer.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 (PEG 300 only, right flank)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 (PEG 300 only, right flank)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test item at 10% in PEG 300 (left fank)

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test item at 10% in PEG 300 (left flank)

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

alpha-Hexylcinnamaldehyde 1% in PEG 300 (right cranial flank)

No. with + reactions:

7

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

alpha-Hexylcinnamaldehyde 1% in PEG 300 (right cranial flank)

No. with + reactions:

4

Total no. in group:

10

Readings and Scoring

The scoring system was performed by visual scoring of erythema, oedema and other clinical changes of skin conditions. They were assessed using the following Magnusson and Kligman grading scale:

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

Grading of all animals was done by positioning the animal under true-light (Philips TLD 36W/84 or Osram 36W/31 830).

Results of the main study

Skin effects after intradermal induction - performed on test day 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

Skin effects after epidermal induction - performed on test day 8

Control group:

No erythematous or oedematous reaction was observed in the animals treated with PEG 300 only.

Test group:

Discrete/patchy erythema was observed in two animals at the 24- and 48-hour reading after treatment with the test item at 15 % in PEG 300.

Skin effects after the challenge - performed on test day 22

Control group:

No skin reactions were observed in the animals when treated with either PEG 300 only or when treated with the test item at 10 % in PEG 300.

Test group:

Discrete/patchy erythema was observed in eight (at the 24-hour reading) and six (at the 48-hour reading) out of 10 animals when treated with the test item at 10 % in PEG 300.

No skin reactions were observed in the animals when treated with PEG 300 only.

Viability/ Mortality/ Macroscopic findings

There were no deaths during the course of the study, hence no necropsies were performed.

Clinical signs, systemic

No signs of systemic toxicity were observed in the animals.

Body weights:

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the fïndings in the adjuvant sensitisation test in guinea pigs the test substance is judged to be a skin sensitizer.

Executive summary:

In order to assess the cutaneous allergenic potential of the test item, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/ EEC, B.6.

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 5 % dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 15 % in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epi-dermal application of the test item at 10 % in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and approximately 48 hours after removal of the dressing.

No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred.

Eight (at the 24-hour reading) and six (at the 48-hour reading) out of 10 test animals showed discrete/patchy erythema after the challenge treatment with the test item at 10 % (w/w) in PEG 300. No skin effect was observed in the control group.

Based on the fïndings in the adjuvant sensitisation test in guinea pigs the test substance is judged to be a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

In order to assess the cutaneous allergenic potential of the test item, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/ EEC, B.6 (BASF, 2006).

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 5 % dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 15 % in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epi-dermal application of the test item at 10 % in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and approximately 48 hours after removal of the dressing.

No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred.

Eight (at the 24-hour reading) and six (at the 48-hour reading) out of 10 test animals showed discrete/patchy erythema after the challenge treatment with the test item at 10 % (w/w) in PEG 300. No skin effect was observed in the control group.

Based on the fïndings in the adjuvant sensitisation test in guinea pigs the test substance is judged to be a skin sensitizer.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is to be classified with category 1B, H317 (May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

Further, the registration substance contains maleic anhydride as an impurity ≥ 1% and is therefore to be classified as Resp. Senis. 1, H334 according to Regulation (EC) No 1272/2008.