Reaction mass of bis(2-ethylhexyl) adipate and cyclohexyl 2-ethylhexyl adipate and dicyclohexyl adipate

Endpoint summary

Description of key information

GPMT: negative (BASF 2019)

LLNA: inconclusive (BASF 2018)

 

 

The skin sensitization potential of the test item Reaction mass of Hexanedioic acid, 1 -cyclohexyl 6-(2-ethylhexyl) ester, Hexanedioic acid, 1,6-dicyclohexyl ester and Hexanedioicacid, 1,6-bis (2-ethylhexyl) ester was examined based onthemethods of the OECD guideline 406 (Guinea Pig Maximization test GMPT).

Altogether, two main tests were accomplished with 30 guinea pigs being organized in twocontrol groups of each 5 animals and two test groups of each 10 animals. The test concentrations for the main tests were determined according to the results of preliminary investigations (dermal and intradermal pre-test).

The main tests started with a 2-week induction period in which the animals of the test groupwere induced with the test item where as animals of the control group were induced with the vehicle (liquid paraffin). In the first induction, the 5%(w/w) test item in the vehicle liquid paraffin and in an adjuvant solution [1:1-mixture of Freund's Complete Adjuvant (FCA) and NaCI 0.9%] was intradermal injected in pairs into the scapular area of the animals of the test group. One week after intradermal induction, the 100% (undiluted) test item was applied underocclusive conditions on the skin of the scapular area of the animals of the test group for 48 hours.

Three weeks after the first induction, animals of the control and test group in both main tests were subjected to the same challenge treatment comprising a 24-hour occlusive applicationof the 75 % (w/w) test item onto the skin of the caudal right flank as well as occlusive application of the vehicle liquid paraffin onto the skin of the caudal left flank.Skin reactions wereassessed 24 and 48 hours after patch removal.

In the first main test, 2 of the 10 animals of the test group (20%, 2/10) and 1 of the 5 animals of the control group (20 %, 1/5) responded with a discrete or patchy erythema (grade 1) to the challenge treatment with the test item 48 hours post application.In a second main test with 15 additional animals this outcome should be confirmed.

In the second main test, none of the 10 animals of the test group (0 %, 0/10) responded with any skin reaction to the challenge treatment with the test item neither 24 nor 48 hours post applicationem.In the control group, 1 of the 5 animals (20 %, 1/5) displayed a discrete orpatchy erythema (grade 1) 24 and 48 hours after the challenge treatment with the test item. Since no significant differences between the control and test groups were observed and the threshold value of 30 % was not exceeded no rechallenge was performed.

Results of the Guinea Pig Maximization Test (GPMT) described in this final report did not provide sufficient evidence for a skin sensitizing potential of the test item Reaction mass of Hexanedioic acid, 1-cyclohexyl 6-(2-ethylhexyl) ester, Hexanedioic acid, 1,6-dicyclohexyl ester and Hexanedioic acid, 1,6-bis (2-ethylhexyl)ester).

 

In an LLNA (BASF 2018), the test substance applied undiluted induced a positive result, 50% application of the test-substance preparation lay just above the cutoff values.

However, the undiluted test-substance caused statistically significant increases in ear weights exceeding the cutoff value (SI ≥ 1.25), and therefore demonstrated excessive ear skin irritation. The observation of slight scaling in two animals of the high dose test group (undiluted) further underpins this finding. The 50% concentration also elicited statistically significant and evident increases in ear weights (mean SI 1.17). The increase of ear weight of the 10% concentration was statistically significant but not relevant.

Therefore, it remains unclear, whether the lymph node responses observed were due to skin sensitizing properties of the test substance or have to be attributed to ear skin irritation.

Thus, a GMPT (Guinea Pig Maximation test) was conducted, since the scapular region of guinea pigs was considered to be less sensitive to a potential irritating potential.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

It was not clear if the results obtained in the LLNA originated from an irritant potency at the ear. Thus, a GMPT (Guinea Pig Maximation test) was conducted, since the scapular region of guinea pigs was considered to be less sensitive to a potential irritating potential.

Species:

guinea pig

Strain:

Dunkin-Hartley

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH
- Housing: 2-3 animals per cage
- Diet (e.g. ad libitum): Altromin 3123" (Altromin, D-32791 Lage, Lippe (ad libitum)
- Water (e.g. ad libitum): domestic quality drinking water being enriched with vitamin C and acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3 °C,
- Humidity (%): 30 - 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

other: liquid paraffin

Concentration / amount:

5% (w/w)

Route:

other: dermal

Vehicle:

other: liquid paraffin

Concentration / amount:

100%

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: liquid paraffin

Concentration / amount:

75%

No. of animals per dose:

Preliminary study: 4 animals
Control group: 5 animals
Test group: 10 animals

Details on study design:

lntradermal pre-test:
The intradermal pre-test was accomplished with one test concentration in 2 guinea pigs as subsequently described. Generally, the intradermal pre-test served for concentration determination of the test item to be applied in the main test. Therefore, a concentration for intradermal induction has to be determined which is not causing skin necrosis 24 and 48 hours post applicationem.
Dermal pre-test:
The dermal pre-test was accomplished with 4 guinea pigs as subsequently described. Generally, the 4 animals were treated with adjuvant (FCA) 3 to 4 weeks before conduction of the dermal pre-test. Therefore, animals were shaved in the scapular region and pairwise injected in two rows of 4 intradermal injections of 0.1 ml of adjuvant. The dermal pre-test served for concentration determination of the test item to be applied in the main test. For that, the minimal irritating concentration for dermal induction and the maximal non-irritating concentration for challenge have to be determined.

Main test: Induction and challenge
Day -1 - Shave of the scapular region (approx. 4 x 6 cm) with an electric hair trimmer and razor
Day 0 - Group-specific setting of three pairs of intradermal injections (dose volume 0.1 ml)
Day 1 and 6 - Examination of intradermal injection sites for signs of skin irritations
Day 6 - Elicitation of a mild irritation by using sodium lauryl sulphate in case that the test item is not skin irritant
- Shave of the injection sites with an electric hair trimmer
- Massage of about 0.5 g sodium lauryl sulphate (10 % in Vaseline) in the skin
Day 7 - dermal induction in scapular region:
- Shave of scapular region, in case that this was not done already on day 6
- Test group: complete loading (approx. 0.5 g or 0.5 ml) of patch (made of filter paper, 2 x 4 cm) with the test item in the minimal irritant concentration
- Control group: complete loading (0.5 ml) of a patch (made of filter paper, 2 x 4 cm) with the vehicle
- Application of this patch on the shaved skin; Occlusive covering of the patch with impermeable adhesive tape (Blenderm®); Fixation of the patch with Micropor® or Gothaplast® adhesive tape (width 5 cm) by wrapping the trunks of the animals; 48-hour exposure time
Day 9 - Removal of the bandage and patch after the 48-hour exposure time
- Examination of the application sites with regard to signs of skin irritation
Day 20 - Shave of the posterior flanks on both sides (approx. 4 x 4 cm) with an electric hair trimmer and razor
Day 21 - topical challenge application in the flank region:
- Loading (approx. 0.5 g or 0.5 ml) of a patch (made of multi-layered gauze, 2 x 2 cm) with the test item in the maximal non-irritant concentration and application of this patch on the posterior right flank of the animals of the test and control group
- Loading (0.5 ml) of a patch (made of multi-layered gauze, 2 x 2 cm) with the vehicle and application of this patch on the posterior left flank of the animals of the test and control group; Application of the vehicle can be omitted, if the vehicle is water
- Occlusive covering of both patches with impermeable adhesive tape (Blenderm®)
- Fixation of both patches with Micorpor® or Gothaplast® adhesive tape (width 5 cm) by wrapping the trunk of the animals
- 24-hour exposure time
Day 22 - Removal of the bandage and patch after the 24-hour exposure time and cleansing of the skin with water under addition of mild soap (e.g. Baktolin®, company Bode Chemie); possible residues of glue from the tape can be removed from the skin with a 1 : 1-mixture of PEG and water
Day 23 - Shave of the posterior flank regions with an electric hair trimmer at least 3 hours before the reading of skin reactions
- Cleansing of skin areas, if necessary, in order to facilitate the assessment
Day 23 and 24 - Examination of each skin area 24 and 48 hours after the termination of the challenge application and assessment of skin reactions

Challenge controls:

The control animals were treated as described above.

Positive control substance(s):

yes

Remarks:

a-Hexylcinnamaldehyde

Positive control results:

The last positive control study with the reference material a-Hexylcinnamaldehyde, technical grade, 85 % was conducted from October to November 2018. In the test group, 10 of 10 guinea pigs (100 %) responded with positive skin reactions.

Reading:

1st reading

Hours after challenge:

24

Group:

other: test group (1st main test)

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

other: test group (1st main test)

Dose level:

75%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: in one animal with scaling

Remarks:

(grade 1, discrete or patchy erythema)

Reading:

2nd reading

Hours after challenge:

48

Group:

other: control group (1st main test)

Dose level:

75%

No. with + reactions:

1

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

24

Group:

other: test group (2nd main test)

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

other: test group (2nd main test)

Dose level:

75%

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

other: control group (2nd main test)

Dose level:

75%

No. with + reactions:

1

Total no. in group:

5

Intradermal and dermal induction:

On day 0, intradermal induction was conducted according to chapter 9.3.3. In the scapular region of the animals, 3 pairwise arranged intradermal injections were applied containing the following preparations:

Control group:

Injection (1) - 1: 1-mixture (v/v) of FCA and NaCI 0.9 %

Injection (2) - vehicle liquid paraffin

Injection (3) - 50 % (w/v) preparation of the vehicle liquid paraffin in a 1: 1-mixture (v/v) of

FCA and NaCI 0.9 %

Test group:

Injection (1)- 1:1-mixture (v/v) of FCA and NaCI 0.9 %

Injection (2) - 5 % (w/w) test item in the vehicle liquid paraffin

Injection (3) - 5 % (w/w) test item in a 1: 1-mixture (v/v) of FCA and NaCI 0.9 %

On day 7, dermal induction was conducted. In the scapular region of the animals, a patch was applied loaded with the following preparation for 48 hours:

Control group: patch with 0.5 ml of the vehicle liquid paraffin

Test group: patch with 0.5 ml of the 100 % test item

In all animals of the two test groups and two control groups, intradermal injections of the 1: 1 -mixture of FCA and NaCI 0.9 % (v/v) evoked skin reactions of grade 3 (intense erythema and swelling) on day 1 post applicationem. The injection sites were necrotic and partially open/bloody or respectively scurfy on day 6. Necrotic injection sites being open/bloody were observed on day 9.

Skin areas which were treated intradermal with the test item prepared in the vehicle displayed a discrete or patchy erythema (grade 1) in majority of animals of the test group on day 1 post applicationem. The most injection sites were free of reactions (grade 0) on day 6 and 9 post app/icationem. Some skin areas of the control groups in which the vehicle was injectted intradermal displayed a discrete or patchy erythema (grade 1) on day 1 post applicationem. A discrete or patchy erythema (grade 1) was also observed on day 6 post app/icationem in most animals of the control groups, whereas all control group animals were free of any reactions (g rade 0) on day 9 post applicationem.

Both the intradermal injections with the test item in the adjuvant as weil as the intradermal injections with the vehicle in the adjuvant caused group-specifically skin reactions of grade 2 (moderate and confluent erythema) in all animals of the test and control groups on day 1 post applicationem. The involved skin areas were moderately red (grade 2) in the control groups on day 6 and in the test groups necrotic and scurfy as weil as partially open/bloody. Formation of eschar was observed in the control groups on day 9, whereas the skin areas were necrotic and open/bloody in the test groups.

Challenge:

On day 21, challenge treatment was performed, whereby one patch with the 75 % (w/w) test item was applied on the caudal right flank and one patch with the vehicle liquid paraffin was applied on the caudal left flank of all animals of the test group and the control group for 24 hours.

During the first main test, a discrete or patchy erythema (grade 1) was observed in 20 % of the animals of both the control group (1/5) and the test group (2/10) after 48 hours.

Skin reactions after the challenge treatment with the test item in the second main test:

Skin reactions		Control group		Test group
		24 hours	48 hours	24 hours	48 hours
Grade 0	No visible change	5/5	4/5	10/10	8/10
Grade 1	Discrete or patchy erythema	0/5	1/5	0/10	2*/10
Grade 2	Moderate and confluent erythema	0/5	0/5	0/10	0/10
Grade 3	Intense erythema and swelling	0/5	0/5	0/10	0/10

* in one animal with scaling

During the second main test, 1 of 5 control group animals displayed a discrete or patchy erythema of grade 1 after 24 and 48 hours. In the test group, no skin reactions were seen.

Skin reactions after the challenge treatment with the test item in the second main test:

Skin reactions		Control group		Test group
		24 hours	48 hours	24 hours	48 hours
Grade 0	No visible change	4/5	4/5	10/10	10/10
Grade 1	Discrete or patchy erythema	1/5	1/5	0/10	0/10
Grade 2	Moderate and confluent erythema	0/5	0/5	0/10	0/10
Grade 3	Intense erythema and swelling	0/5	0/5	0/10	0/10

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the Guinea Pig Maximization Test (GPMT), the test item Reaction mass of Hexanedioic acid, 1-cyclohexyl 6-(2-ethylhexyl) ester, Hexanedioic acid, 1,6-dicyclohexyl ester and Hexanedioic acid, 1,6-bis (2-ethylhexyl) ester has no skin sensitizing potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item was not classified and labelled accordingto Regulation (EC) No 1272/2008 (CLP).