Reaction products of N-Phenyl-1-Naphthylamine with Nonene, branched

Endpoint summary

Description of key information

GPMT (2021): negative

cLLNA (2022): negative

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17.07.92

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The LLNA gave questionable results. Thus, a GPMT was performed to clarify the LLNA results. This GPMT together with the additionally performed cLLNA confirm that the LLNA is not a suitable testsystem for this chemical.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH
- Weight at study initiation: 300-340 g
- Housing: 2 to 3 animals per page
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

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

Route:

intradermal and epicutaneous

Vehicle:

other: liquid paraffin

Concentration / amount:

intradermally: 5 %
dermal: 100 %

Day(s)/duration:

intradermally: once; dermally: day 7 to day 9

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100 % 1st test

Day(s)/duration:

day 21 to day 22

Adequacy of challenge:

other: not adequte as 80 % of control animals showed irritation

No.:

#2

Route:

epicutaneous, semiocclusive

Vehicle:

other: liquid paraffin

Concentration / amount:

75 % 1st test (re-challenge)

Day(s)/duration:

day 28 to day 29

Adequacy of challenge:

other: irritation in 20 % of the animals

No.:

#3

Route:

epicutaneous, semiocclusive

Vehicle:

other: liquid paraffin

Concentration / amount:

75 % 2nd test

Day(s)/duration:

day 22 to day 23

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10

Details on study design:

Intradermal 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 deter-mination of the test item to be applied in the main test. Therefore, a concentration for intradermal induction had to be determined without causing skin necrosis 24 and 48 hours post applicationem.

Day -1 – shave
Shave of two animals in the scapular region (approx. 4 x 6 cm) within an electric hair trimmer and razor
Day 0 – application
Intradermal injection of 0.1 mL each in pairs and three rows in the scapular region (approx. 2 x 4 cm) of two animals each according to the scheme in Table 3
Arrangement of injection pairs during the intradermal pre-test.
Position Injection pair Composition of injection solution
cranial 1 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 %
central 2 Test item in the vehicle in the selected concentration
caudal 3 Test item in the selected concentration in a 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 %

Day 1 and 2 – observation of injection sites
Observation of intradermal injection sites 24 and 48 hours post applicationem; assessment of results according to chapter 9.3.4. (Table 5)

Dermal pre-test

The dermal pre-test was accomplished with 3 guinea pigs as subsequently described. Generally, the 3 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.

Day -29 or -22 – shave
Shave of three animals in the scapular region (approx. 4 x 6 cm) with an electric hair trimmer as well as razor
Day -28 or -21 – intradermal injection of FCA
Intradermal injections of 0.1 mL each of 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 % in pairs and two rows in the scapular region (approx. 2 x 2 cm) of three animals
Day -1 – shave
Shave of three animals on both sides (left and right) in the flank regions (cranial and caudal) with an electric hair trimmer and razor
Day 0 – topical application in the flank region
Loading (approx. 0.5 g or 0.5 mL per patch) of four patches (made of multi-layered gauze, 2 x 2 cm) with the test item in the selected concentration and application of these patches on the flanks of animals in a sequence which begins with the highest concentration on the cranial flank of the left side and ends with the lowest concentration on the caudal flank of the right side
Occlusive covering of the patches with impermeable adhesive tape (Blenderm®)
Fixation of patches with Micropor® or Gothaplast® adhesive tape (width 5 cm) by wrapping the trunk of animals
24-hour exposure time
Day 1 – removal of patch and skin cleansing
Removal of the bandage and patches after 24-hour exposure time; cleansing of skin lonely with water and mild soap (e.g. Baktolin®; company Bode Chemie) was not possible due to partial stickiness and viscosity of TI preparation; but residues of glue from the tape and test item could be removed carefully with 1:1-mixture of PEG (polyethylene glycol) and water
Day 2 and 3 – observation of injection sites
Observation of dermal application sites 24 and 48 hours post applicationem; assess¬ment of the results according to chapter 9.3.4. (Table 5)

Main test with induction and challenge as well as re-challenge

Day -1 – shave
Shave of the scapular region (approx. 4 x 6 cm) with an electric hair trimmer and razor
Day 0 – intradermal induction
Group-specific setting of three pairs of intradermal injections (dose volume 0.1 mL) in the prepared test field of the scapular region (Table 4)

Group-specific arrangement of the injection pairs during the intradermal induction phase.
TEST GROUP
Position Injection pair Composition of injection solution
cranial 1 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 %
central 2 Test item in the vehicle in the selected concentration
caudal 3 Test item in the selected concentration in a 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 %
CONTROL GROUP
Position Injection pair Composition of injection solution
cranial 1 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 %
central 2 Vehicle
caudal 3 50 % (w/v) preparation of the vehicle in a 1:1-mixture (v/v) of FCA and aqua ad inj. or NaCl 0.9 %

Day 1 and 6 – observation of injection sites
Examination of intradermal injection sites for signs of skin irritations; assess¬ment of the results according to chapter 9.3.4. (Table 5)
Day 6 – treatment with sodium dodecyl sulphate
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 patch and observation of the application site
Removal of the bandage and patch after the 48-hour exposure time
Examination of the application sites with regard to signs of skin irritation; assess¬ment of the results according to chapter 9.3.4. (Table 5)
Day 20 – shave
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 patch and skin cleansing
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 – preparing of the treated areas
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 – observation of skin reactions
Examination of each skin area 24 and 48 hours after the termination of the challenge application and assessment of skin reactions according to chapter 9.3.4. (Table 5)
Day 27 – shave
Shave of the anterior flanks on both sides (approx. 4 x 4 cm) with an electric hair trimmer and razor
Day 28 – topical re-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 chosen concentration and application of this patch on the anterior left 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 anterior right 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 29 – removal of patch and skin cleansing
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 30 – preparing of the treated areas
Shave of the anterior 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 30 and 31 – observation of skin reactions
Examination of each skin area 24 and 48 hours after the termination of the re-challenge application and assessment of skin reactions according to chapter 9.3.4. (Table 5)

Assessment of skin reactions
Skin reactions were assessed according to the grading scale by Magnusson/Kligman (Table 5). In case of difficult or uncertain results, assessment was conducted with a manual inspec-tion lamp having white light and twofold magnification.
 

Grading scale according to Magnusson/Kligman.
Skin reaction Grade
no visible change 0
discrete or patchy erythema 1
moderate and confluent erythema 2
intense erythema and swelling 3

Positive control substance(s):

not required

Remarks:

The last positive control study (Lab. No. 04289) with the reference material α-Hexylcinnam-aldehyde (purity: 85 %) was conducted from March to June 2021. In the test group, 80 % of the guine pigs responded with positive skin reactions to the treatment.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100 % 1st main test

No. with + reactions:

4

Total no. in group:

5

Remarks on result:

not determinable

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100 % 1st main test

No. with + reactions:

2

Total no. in group:

5

Remarks on result:

not determinable

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 % 1st main test

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

not determinable

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 % 1st main test

No. with + reactions:

3

Total no. in group:

10

Remarks on result:

not determinable

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

75 % 1st main test

No. with + reactions:

1

Total no. in group:

5

Remarks on result:

not determinable

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

75 % 1st main test

No. with + reactions:

1

Total no. in group:

5

Remarks on result:

not determinable

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

75 % 1st main test

No. with + reactions:

5

Total no. in group:

10

Remarks on result:

not determinable

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

75 % 1st main test

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

not determinable

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

75 % 2nd main test

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:

75 % 2nd main test

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 % 2nd main test

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75 % 2nd main test

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Main tests

Intradermal and dermal induction

On day 0, intradermal induction was conducted according to chapter9.3.3. 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 NaCl 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 NaCl 0.9 %

Test group

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

On day 7, dermal induction was conducted according to chapter9.3.3. 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 NaCl 0.9 % (v/v) evoked skin reactions grade 2 at least on day 1post applicationem. The injection sites were necrotic and open/bloody on days 6 and 9.

Skin areas which were treated intradermally with the test item prepared in the vehicle dis­played a discrete or patchy erythema (grade 1) in majority of animals of the two test groups on day 1 post applicationem. A discrete or patchy erythema (grade 1) was also still observed in half of the animals on day 6post applicationem, whereas the other half of animals was already free of reactions (grade 0) on this day. No skin reactions (grade 0) were observed on day 9 post applicationem or assessment of erythema formation was prevented by rusty-red coloration of the TI-treated skin and adjacent hair. Most of the skin areas of the control groups in which the vehicle was inject­ed intra­der­mally 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 6post applica­tionemin 50 % of the control group animals, whereas the majority of control group ani­mals was free of any reactions (grade 0) on day 9 post applicationem.

The intradermal injections with the test item in the adjuvant mixture caused mostly skin reactions of grade 2 (moderate and confluent erythema) in the test group animals on day 1post applicationemas well as bloody/open necrosis on days 6 and 9 post applicationem. Also, the majority of the control group animals displayed a moderate and confluent erythema (grade 2) on day 1post applicationemand necrosis on day 6 post applicationem. Moreover, eschar formation was observed in the control group animals on day 9 post applicationembeside the necrosis.

Challenge

 

On day 21, challenge treatment was performed according to chapter9.3.3., whereby one patch with the test item preparation 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 groups and the control groups for 24 hours.

The difference between the challenge treatments in the 1^stand 2^ndmain tests was the TI concentration which was applied. Whereas the undiluted test item was used for the 1^stmain test, the TI preparation for the 2^ndmain test had a reduced concentration of 75 %w/w.

This alteration was also the reason for differing findings which were observed in 1^stand 2^ndmain test as responses to the challenge treatments with different concentrations of the test item.

During the 1^stmain test, skin reactions ranging between grades 1 and 2 were observed in 80 % (4/5) of the control group animals and in 90 % (9/10) of the test group animals 24 hours after the challenge treatment with the 100 %w/w test item. The incidence of skin reactions decreased to 40 % (2/5) in the control group and to 30 % (3/10) in the test group after 48 hours and also intensity of erythema decreased over time as presented inTable9.

These findings corroborated the suspicion of a mechanical irritation effect concluded from the difficulties to remove the patch and TI residues at the end of the exposure time, because these were stuck intensely to the skin. In spite of soaking the dressing with PEG/water-mixture, the TI-treated skin was reddened and displayed partially micro-in­juries at the end of the challenge treatment.

During the 2^ndmain test, 2 of 10 (20 %) test group animals displayed a discrete or patchy ery­thema of grade 1 and scales after the challenge treatment with the 75 %w/w test item, whereas the control group was free of any skin reaction as presented inTable10.

The patch removal at the end of the exposure time was difficult again and required intense and time-consuming soaking of the dressing with soap-water and PEG/water-mixture.

Re-challenge

 

The ambiguous challenge results of the 1^stmain test under involvement of a mechanical irritation effect caused by the problematic stickiness of the test item required the conduction of a re-challenge one week later with reduced TI concentration and the same animals of the control group 1 and the test group 1.

On day 28, re-challenge treatment was performed during the 1^stmain test according to chapter9.3.3., whereby one patch with the 75 %w/w test item was applied on the cranial left flank and one patch with the vehicle liquid paraffin was applied on the cranial right flank of all animals of the test group 1 and the control group 1 for 24 hours.

Adhesive property of TI preparation complicated again the patch removal at the end of exposure time, but intense soaking of dressings with soap-water and PEG/water mixture allowed finally detachment without obvious micro lesions.

A discrete or patchy erythema (grade 1) was observed in 20 % (1/5) of the control group animals and in 30 % (3/10) of the test group animals after the re-challenge treatment with the 75 %w/w test item. Moreover, 2 of 10 (20 %) test group animals displayed a moderate and confluent erythema of grade 2 after 24 hours which only may be considered as positive skin reaction. A summary of findings is presented inTable11.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the Guinea Pig Maximization Test (GPMT) described in this Final Report, the test item XPDL 958 shows no skin sensi¬tizing potential under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 22, 2010

Deviations:

yes

Remarks:

The animals are not sacrificed on day 6 and are kept for at least 16 days. After this period the “primed” animals are treated only once with the test item (challenge phase) and the secondary immune response is quantified analogous to the LLNA (OECD 429).

Principles of method if other than guideline:

In an LLNA assay (58V0205/20B048) the test item has been previously described to induce increases of 3H-thymidine (mean SIs of 5.81, 5.53 and 6.61) without significant increases in ear weights (mean SIs of 1.04, 1.04 and 1.07). There was no concentration dependence of the effects.
The LLNA quantitively determines the exposure level required for initiation of a primary immune response (sensitization phase). This study aims at addressing the level required for the induction of a secondary immune response (elicitation threshold) in already sensitized animals.
For this purpose, a modified version of the LLNA (challenge LLNA (cLLNA)) was applied. The protocol of the cLLNA resembles the LLNA in the induction phase (sensitization phase). Groups of animals are treated on 3 consecutive days with a single dose of the test item. In contrast to the LLNA, however, the animals are not sacrificed on day 6 and are kept for at least 16 days.
This allows the induced primary response to completely take place and disappear. The only parameter remaining from the primary immune response should be the silent memory T-lymphocytes, which are responsible for the induction of a secondary immune response.
After this period the “primed” animals are treated only once with the test item (challenge phase) and the secondary immune response is quantified analogous to the LLNA (OECD 429) via measurement of the lymphocyte proliferation.
The determination of the elicitation threshold in the challenge phase is achieved by treating different groups of primed animals with decreasing doses of the test item. Appropriate control groups are required for determination of the stimulation indices and exclusion of irritation effects (challenge control group).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd / SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH; Kreuzelweg 53; NL-5961 NM Horst
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16.3 g – 21.1 g (pretest); 16.8 g – 19.9 g (main test)
- Housing: 1 animal per cage
As group housing may increase oral exposure due to grooming of the animals and may interfere with clear observations of each individual animal, animals were single housed for the duration of the test.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before the first test substance application
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-65 %
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

methyl ethyl ketone

Concentration:

Induction: three times with a 25% (w/w) preparation of the test substance in MEK
Challenge: once with 2.5%, 5%, 10% and 25% (w/w) preparations of the test substance in MEK or with the vehicle alone

No. of animals per dose:

5

Details on study design:

OBJECTIVES
In an LLNA assay (58V0205/20B048) the test item has been previously described to induce increases of 3H-thymidine (mean SIs of 5.81, 5.53 and 6.61) without significant increases in ear weights (mean SIs of 1.04, 1.04 and 1.07). There was no concentration dependence of the effects.
The LLNA quantitively determines the exposure level required for initiation of a primary immune response (sensitization phase). This study aims at addressing the level required for the induction of a secondary immune response (elicitation threshold) in already sensitized animals.
For this purpose, a modified version of the LLNA (challenge LLNA (cLLNA)) was applied. The protocol of the cLLNA resembles the LLNA in the induction phase (sensitization phase). Groups of animals are treated on 3 consecutive days with a single dose of the test item. In contrast to the LLNA, however, the animals are not sacrificed on day 6 and are kept for at least 16 days. This allows the induced primary response to completely take place and disappear. The only parameter remaining from the primary immune response should be the silent memory T-lymphocytes, which are responsible for the induction of a secondary immune response.
After this period the “primed” animals are treated only once with the test item (challenge phase) and the secondary immune response is quantified analogous to the LLNA (OECD 429) via measurement of the lymphocyte proliferation.
The determination of the elicitation threshold in the challenge phase is achieved by treating different groups of primed animals with decreasing doses of the test item. Appropriate control groups are required for determination of the stimulation indices and exclusion of irritation effects (challenge control group).

SELECTION OF DOSES/CONCENTRATIONS AND VEHICLE
In a previously performed LLNA according to OECD TG 429 (see study 58V0205/20B048) methyl ethyl ketone (MEK) was used as vehicle because good solubility/homogeneity of the preparation was achieved. Therefore, this vehicle was also used for the present study.
In the LLNA (see study 58V0205/20B048) it could be shown that treatment of the animals with concentrations of 25%, 50% and the undiluted test substance did not cause irritation as determined by measuring the ear weight (stimulation indices 1.04, 1.04 and 1.07) in parallel to the increased lymphocyte proliferation (3H-thymidine incorporation stimulation indices of 5.81, 5.53 and 6.61).
There was no concentration dependence of the effects.
In a non-GLP pre-experiment to the cLLNA (with another batch of the test substance, 20/0205-4) testing test-substance-preparations up to 25%, no relevant lymphocyte proliferation was observed after a single application of the 25% test-substance preparation in MEK (stimulation index thymidine incorporation 1.43, stimulation index cell counts 1.34) without relevant increase in ear weight.
Thus, the highest concentration, which could be used for induction without provoking unspecific reactions (thymidine incorporation) was 25%.
In this study the test item was used as a 25% solution in MEK for the sensitization phase and as 25%, 10%, 5% and 2.5% solutions for the challenge phase.
Treatment scheme see table in "any other information on materials and methods incl. tables".

TEST-SUBSTANCE PREPARATION
Test-substance preparation: The test-substance preparation was produced on a weight per weight (w/w) basis shortly before application. After stirring with a magnetic stirrer, the test substance was soluble in the vehicle.
Vehicle: MEK (methyl ethyl ketone)
Reason for the vehicle: MEK was used as the vehicle because good solubility of the preparations was achieved.
Form of application: Solution

ANALYSES
Stability of the testsubstance preparation: Because the test-substance preparations were applied shortly after preparation, no analysis of the test substance in the vehicle was required.
Homogeneity of the testsubstance preparation: No homogeneity analysis was carried out because the testsubstance preparations are solutions.
Concentration control analysis of the testsubstance preparation: No concentration control analysis was carried out.
Food analysis: The food used in the study will be assayed for chemical and microbial contaminants. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p 27354 (EPA), will serve as the guideline for maximum tolerable contaminants. Additionally, the levels of phytoestrogens should not exceed 350 μg of genistein equivalents/gram. According to recommendations of the GVSOLAS, the total amount of bacteria must not exceed 1*10^5 per g food.
Drinking water analysis: The drinking water is assayed regularly for chemical contaminants both by the municipal authorities of Frankenthal, Germany and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation serves as the guideline for maximum tolerable contaminants.
Bedding analysis: The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). The values given in Lab Animal, Nov.–Dec. 1979, pp 24–34, serve as the guideline for maximum tolerable contaminants.
Enrichment (nest-building material) analysis: The enrichment (nest-building material) is assayed regularly for contaminants (chlorinated hydrocarbons and heavy metals).

EXPERIMENTAL PROCEDURE
Conduct of the study
The study comprised four treatment groups, one vehicle control group and one challenge control group. Each group consisted of 5 mice.
Randomization: Prior to first application, the animals will be distributed to the individual groups, will receive their animal numbers and will be allocated to the respective cages according to the randomization instructions of WinRando Version 3.2.
Body weight determination: Individual body weights on day 1 prior to the first application and on day 23 prior to the sacrifice of the animals.
Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal part of both ears
Frequency of application: 3 consecutive applications (study day 1 – study day 3) to the same application site. Single challenge treatment on study day 21.
The animals of vehicle control group 0, challenge control group 1 and test groups 2 - 5 were treated with the vehicle or a test-substance preparation.

³H thymidine injection
On study day 23 ca. 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.

Terminal procedures
The animals were sacrificed on study day 23 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
Determination of ear weight: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using a PC-based tabular calculation software. The mean and individual data were not always rounded, but the significant digits were produced by changing the display format.
Consequently, calculation of mean values by using the individual data presented in the report will in some instances yield minor variations in value.

Calculations and statistical analyses
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group or challenge control group.

Statistical test and parameter
3H-thymidine incorporation, cell count, lymph node weight and ear weight: WILCOXON - Test

EVALUATION OF RESULTS
A relevant sensitization effect is determined by the comparison of the 3H-thymidine incorporation levels and cell count levels of the test-substance treated groups to the vehicle control group and challenge control group.
The challenge control group distinguishes between irritation and sensitization effects of the test substance.

Positive control substance(s):

not specified

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Parameter:

SI

Value:

1.61

Test group / Remarks:

challenge control

Parameter:

SI

Value:

1.44

Test group / Remarks:

induction: 25 %, challenge: 25 %

Parameter:

SI

Value:

1.43

Test group / Remarks:

induction: 25 %, challenge: 10 %

Parameter:

SI

Value:

1.54

Test group / Remarks:

induction: 25 %, challenge: 5 %

Parameter:

SI

Value:

1.28

Test group / Remarks:

induction: 25 %, challenge: 2.5 %

CLINICAL OBSERVATIONS AND ASSESSMENT OF FINDINGS

Pretest / Irritation Screening

In the pretest with another batch of the test substance (20/0205-4) two mice each were treated once with test-substance concentrations of 2.5%, 5%, 10% and 25%.

No signs of systemic toxicity were observed in the pretest.

At the tested concentrations, the animals did not show excessive signs of local irritation as confirmed by determination of the ear weights (compared to vehicle values).

After a single application of the 25% test-substance preparation in MEK a stimulation index for 3H-thymidine incorporation of 1.43 and a stimulation index for cell counts of 1.34 was observed.

Main test

3H-thymidine incorporation, cell count and lymph node weight

The 25% concentration applied to the animals during the challenge treatment, which were induced with the vehicle alone (challenge control group) did not induce biologically relevant increases of 3H-thymidine incorporation into the cells and in the auricular lymph node cell counts (no increase above the cutoff Stimulation Index of 3 for 3H-thymidine incorporation and no biologically relevant Stimulation Index of 1.5 for the auricular lymph node cell counts). The increases were statistically significant. In addition, the increase in lymph node weight was statistically significant.

When applied as 25% preparation in MEK (induction treatment) and as 2.5%, 5%, 10% and 25% in MEK (challenge treatment), the test substance did not induce biologically relevant (no increase above the cutoff Stimulation Index of 3), but statistically significant increases of 3Hthymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, no biologically relevant response (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts was observed at all concentrations. The increases were statistically signifant at 2.5%, 5% and 25%.

In addition, statistically significant increases in lymph node weights were noted at the 2.5 and 25% concentration.

For details see attached figures.

Ear weights

The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. The increases were statistically significant at all concentrations.

Body weights

The expected mean body weight gain was generally observed during the study.

Clinical signs

No signs of systemic toxicity were noticed in all animals during general observation.

Local findings

No local findings were observed during the observation period.

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that XPDL 958 does not exhibit a skin sensitizing potential in the Challenge Local Lymph Node Assay (cLLNA) under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

GPMT

The skin sensitization potential of the test item XPDL 958 was examined based on the methods of the OECD guideline 406 (1992)², council regulation (EC) No 440/2008 part B.6. (2008)³and U.S. EPA guideline OPPTS 870.2600 (2003)⁴ in a modified manner. For the study, the Guinea PigMaximization test (GPMT) of MAGNUSSON and KLIGMAN (1970)¹was applied.

Altogether, two main tests were accomplished with 30 guinea pigs being organized in two control 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 group were induced with the test item (XPDL 958) whereas 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 NaCl 0.9 %] was intradermally injected in pairs into the scapular area of the animals of the test group. Animals of the control group were pairwise injected only with the vehicle or a 50 % (w/v) concen­tration of the vehicle in the adjuvant solution in an intradermal manner. Furthermore, all ani­mals got a third pair of intradermal injections consisting of the adjuvant solution as a 1:1-mixture of FCA and NaCl 0.9 %.

One week after intradermal induction, the 100 % (undiluted) test item was applied under occlusive conditions on the skin of the scapular area of the animals of the test group for 48 hours whereas in the control group, dermal induction was accomplished with the vehicle liquid paraffin only.

Following intradermal and dermal induction in the 1^stmain test, all 15 animals of the control and test group displayed graduated skin reactions in form of erythema (grade 1 to 3) or no reactions (grade 0) on the different injection sites. Injections sites involving the adjuvant appeared necrotic and partially open/bloody on the day before the dermal induction. All sites treated with the adjuvant were necrotic and open/bloody or respectively covered with eschar at the end of the dermal in­duction application.

The skin reactions during the 2^ndmain test following intradermal and dermal induction were similar to those from the 1^stmain test and ranged between grades 0 and 2 for ery­the­ma on the different injection sites. Injections sites involving the adjuvant appeared mostly necrotic and open/bloody on the day before the dermal induction. All sites treated with the adjuvant were necrotic and partially open/bloody at the end of the dermal induction application.

Three weeks after the first induction, animals of the control and test group in both main tests were subjected to challenge treatments comprising a 24-hour occlusive application of the 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. However, the challenge treatment was performed with the undiluted test item (100 %) during the 1^stmain test and with the 75 %w/w test item during the 2^ndmain test. Skin reac­tions were always assessed 24 and 48 hours after patch removal.

The sticky-viscous consistency of the test item made removal of patch and test substance residues very difficult despite soaking the patch with water and mild soap or with a 1:1-mixture of water and polyethylene glycol (PEG) respectively. Therefore, micro-injuries were partially observed immediately after peeling off the dressing. This mechanical irritation of TI-treated skin could probably have been one reason for observed skin reactions after the challenge treatment during the 1^stmain test.

In the 1^stmain test, 9 of 10 animals of the test group (90 %, 9/10) responded with skin reac­tions to the challenge treatment with the 100 %w/w test item ranging between grades 1 and 2. Also 4 of 5 control group ani­mals (80 %, 4/5) displayed either a discrete or patchy erythema (grade 1, 2/5) or a moderate and confluent erythema (grade 2, 2/5). The skin areas treated with the vehicle only were free of any skin reactions in both groups. Decrease of response intensity was seen over 48-hour ob­servation time without group-specific differentiation. These findings corroborated the suspicion of involvement of a mechanical irritation effect.

In accordance with the agreement with the sponsor, a re-challenge was performed one week after the challenge during the 1^stmain test. For the re-challenge, the test item was diluted with the vehicle to a concentration of 75 %w/w in order to reduce its adhesive property and the resul­ting mechanical irritancy. The same animals of the control and test group were subjected to the re-challenge treatment comprising a 24-hour occlusive application of the 75 %w/w test item onto the skin of the cranial left flank as well as occlusive application of the vehicle liquid paraffin onto the skin of the cranial right flank. Skin reac­tions were assessed again 24 and 48 hours after a little easier patch removal. Nevertheless, the patches were still slightly adhesive during removal.

During the re-challenge in the 1^stmain test, a discrete or patchy erythema (grade 1) was observed in 3 of 10 test group animals (30 %, 3/10) as well as a moderate and confluent ery­thema (grade 2) in 2 of 10 test group animals (20 %, 2/10). Since 1 of 5 control group ani­mals (20%, 1/5) displayed also a discrete or patchy erythema (grade 1), only the grade-2-responses in the test group (20 %) may be considered as positive skin reactions not passing the threshold value of 30 % for skin sensitizer.

In a 2^ndmain test with 15 additional animals the non-sensitizing outcome of the 1^stmain test should be confirmed under consideration of gained knowledge that reduction of the test item concentration to 75 %w/w facilitates patch removal and assessment of skin reactions without relevant influence of mechanical irritation.

In the 2^ndmain test, 2 of 10 test group animals (20 %, 2/10) responded with a discrete or patchy erythema (grade 1) and scales to the challenge treat­ment with the 75 %w/w test item, whereas the control group was free of any skin reactions.

Performance of a re-challenge was not necessary due to this unambiguous outcome of the challenge in the 2^ndmain test.

The low incidence of positive skin reactions occurring in the two test groups of the 1^stand 2^ndmain test after re-challenge and challenge treatment respectively with the 75 %w/w test item indicated no sensitizing effect of the test item.

Based on the results of the Guinea Pig Maximization Test (GPMT) described in this final report it was concluded that XPDL 958 does not have a sensitizing effect on the skin of the guinea pig under the test conditions chosen.

cLLNA

The LLNA quantitively determines the exposure level required for initiation of a primary immune response (sensitization phase). In the LLNA (see study 58V0205/20B048) it could be shown

that treatment of the animals with concentrations of 25%, 50% and the undiluted test substance XPDL 958 did not cause irritation as determined by measuring the ear weight (stimulation indices 1.04, 1.04 and 1.07) in parallel to the increased lymphocyte proliferation (3H-thymidine incorporation stimulation indices of 5.81, 5.53 and 6.61). There was no concentration dependence of the effects.

The present study, a challenge LLNA, aimed at investigating whether there is a specific secondary immune response and if so at addressing the level required for the induction of a secondary immune response in already sensitized animals.

During the induction treatment groups of 5 female CBA/CaOlaHsd mice each were treated three times with a 25% (w/w) preparation of the test substance in MEK or with the vehicle alone. During the challenge treatment, the animals were treated once with 2.5%, 5%, 10% and 25% (w/w) preparations of the test substance in MEK or with the vehicle alone.

The study used 4 test groups, a vehicle control group and a challenge control group. Each animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle applied to the dorsal surfaces of both ears on three consecutive days (day 1, 2 and 3; induction treatment) or on day 21 (challenge treatment).

Ca. 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice on day 23. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation.

The 25% concentration applied to the animals during the challenge treatment, which were induced with the vehicle alone (challenge control group) did not induce biologically relevant increases of 3H-thymidine incorporation into the cells and in the auricular lymph node cell counts (no increase above the cutoff Stimulation Index of 3 for 3H-thymidine incorporation and no biologically relevant Stimulation Index of 1.5 for the auricular lymph node cell counts). The increases were statistically significant. In addition, the increase in lymph node weight was statistically significant.

When applied as 25% preparation in MEK (induction treatment) and as 2.5%, 5%, 10% and 25% in MEK (challenge treatment), the test substance did not induce biologically relevant (no increase above the cutoff Stimulation Index of 3), but statistically significant increases of 3Hthymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, no biologically relevant response (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts was observed at all concentrations. The increases were statistically signifant at 2.5%, 5% and 25%.

In addition, statistically significant increases in lymph node weights were noted at the 2.5 and 25% concentration.

The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. The increases were statistically significant at all concentrations.

Thus, it is concluded that XPDL 958 does not exhibit a skin sensitizing potential in the Challenge Local Lymph Node Assay (cLLNA) under the test conditions chosen.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Both the available challenge LLNA (cLLNA) and the available GPMT gave a negative result and thus, no classification for skin sensitization is required.

Furthermore, these two tests show, that the conventional LLNA is not a suitable test system for this test substance due to it's high LogPow as there is evidence (see OECD, ENV/CBC/MONO(2021)11/ANN6) that the false-positive rate of the LLNA is higher for lipophilic chemicals. The cLLNA and the GPMT confirm that the cell proliferation in the lymphnode, which was observed in the conventional LLNA without dose-response, has to be considered as unspecific and not related to induction of skin sensitization.