Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Endpoint summary

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:

Start of study: October 25 2001; End of study: February 7 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Remarks:

No deviation that affected the study reliability

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A study was conducted in 2002 according to OECD 406. This study was conducted before suitable In Vitro alternatives were available.

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

Hartley-strain white guinea pigs (48 females) were purchased from Japan Laboratory Animals, Inc. at 5 weeks of age and subjected to quarantine and acclimation to the study environment for 12 days (8 days for the animals used in the preliminary study). During this period, they were observed for general condition every day and weighed at least once a week. Thirty animals that showed regular body weight development without clinical abnormalities were selected and used in the main study. They were at 6 or 7 weeks of age and the body weights ranged from 334 to 431 g on the starting day of induction for senisitzation.

Animals were housed in an animal room in which the temperature was kept at 22.5 ± 3.5°C, relative humidity at 50 ± 20%, air ventilation at 10 to 15 times per hour, and 12-hour lighting per day (from 07:00 to 19:00). The animals were put in stainless wire-mesh cages individually or in groups of 2 animals, and allowed free access to pelleted diet RC4 and water using an automatic watering system.

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

(for injection)

Concentration / amount:

Main Test:
Intradermal induction: 5%
Percutaneous induction: 25%
Challenge exposure: 5%

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

(for injection)

Concentration / amount:

Main Test:
Intradermal induction: 5%
Percutaneous induction: 25%
Challenge exposure: 5%

No. of animals per dose:

Main Test:
Test article sensitization group: 10
Positive control sensitization group: 5
Control group: 5

Details on study design:

PRELIMINARY STUDY TO DETERMINE TO DOSE CONCENTRATIONS OF TEST SOLUTION:
During the quarantine acclimation period, a preliminary study was conducted using some purchased animals to determine the dose concentrations of the test solution for intradermal induction, percutaneous induction and challenge exposure. For intradermal induction, 4 animals with no abnormalities were selected and the test solutions at various concentrations (each 0.1 rnL) administered intradermally to the skin above the scapula where hr was clipped and shaved. For percutaneous application, another 8 animals without abnormalities were selected. Test solution at each dose concentration (0/1 mL) was applied to the patch of 2 x 2 cm and the patch applied to the 8 animals. It was covered with polyethylene film tape for occlusive application. After 24 hours of application, the patches were removed and the application site cleaned using absorbent cotton soaked with water for injection. For both intradermal application and percutaneous application, skin reactions were judged (according to standards of Magnusson & Kligman: 1969) and the dose concentration for each application selected.

The results obtained in the preliminary study are shown below:
Test article BZ-0145B:
For intradermal application, administration was done at 25, 10, 5, 2.5 and 1% to 2 animals. Erythema and edema of score 3 with necrosis were observed at the concentrations of 10% and above. At the concentration of 5% or below, erythema of score 2 was observed in both animals.
For percutaneous application, administration was done at 25, 10, 5, 2.5 and 1% to 4 animals. Erythema of score 1 was observed at the concentrations of 25 and 10%. At the concentration of 5% or below, no skin reactions were observed in any animal.
Based on the results of the preliminary study described above, the following concentrations were selected for the main study: 5%, the maximum irritating concentration without necrosis, for intradermal induction; 25%, the maximum preparable concentration for percutaneous induction; 5%, the maximum non-irritating concentration, for challenge exposure.

MAIN TEST:
GROUP COMPOSITION:
Test groups were composed as follows:
Test article sensitization group (10 animals)
Positive control sensitization group (5 animals)
Control group (5 animals).

DESIGNATION OF STUDY DAYS:
Starting day of induction for sensitization (day of intradermal induction): Day 0 of induction
The day following intradermal induction and thereafter: Day 1 after induction, Day 2 after induction etc.

METHOD OF TREATMENT:
1) SENSITIZATION:
Intradermal induction for sensitization:
On the day before intradermal induction for sensitization, fur of the 4 x 6 cm area on the scapula was clipped using an electric clipper and shaved as short as possible using an electric shaver. On the next day, for all animals in all test groups, 0.1 mL of the substance for intradermal induction (shown in table of 'Substance for Induction' - see any other information on material and methods) was applied to the relevant areas of induction.

Percutaneous induction for sensitization:
Six days after induction for sensitization, the same area was clipped and shaved again, and 0.2 mL of petrolatum containing 10% sodium lauryl sulfate was applied open to the area (2 x 4 cm) for intradermal induction for sensitization for the animals in the positive control sensitization group and the control group. On the next day, the area was cleaned with absorbent cotton soaked with ether, 0.2 mL of the substance for percutaneous induction (shown in table of 'Substance for Induction' - see any other information on material and methods) applied to the area of induction using a patch of 2 x 4 cm, which was covered with polyethylene film tape for 48-hour occlusive application for percutaneous induction of sensitization. For the test article group, sodium lauryl sulfate was not applied since percutaneous induction was done at the concentration causing irritation.
For the control group, water for injection (the vehicle) was used in the same manner as for the sensitization groups.

2) CHALLENGE EXPOSURE:
On Day 20 after induction, the fur of the right and left abdominal flanks (area: 4 x 7 cm) of each animal in each group was clipped and shaved. On the next day, 0.1 mL of the substance for challenge exposure (shown in the table of 'Substance for Challenge' - see any other information on material and methods) was applied to a patch of 2 x 2 cm, which was applied to the application site. It was then covered with polyethylene film tape for 24-hour occlusive application. After the 24-hour application, the patch was removed and the application site cleaned using absorbent cotton soaked with water for injection.

OBSERVATION OF GENERAL CONDITION:
The animals were observed for general condition once a day from the starting day of induction for sensitization (Day 0) to the ending day of skin observation after challenge exposure (Day 24).

MEASUREMENT OF BODY WEIGHT:
The animals were weighed on the starting day of induction for sensitization (Day 0), day of percutaneous induction (Day 7), day of challenge exposure (Day 21) and ending day of observation (Day 24).

OBSERVATION OF SKIN REACTIONS:
Skin reactions were observed at 24 and 48 hours after removal of application for challenge exposure and the reactions observed were scored according the the evaluation table of skin reactions below:

Severity of skin reactions: Score:
No gross change 0
Sporadic or macular erythema 1
Moderate or diffuse erythema 2
Strong erythema and edema 3

After the end of the observation, the animals were euthanized by deep ether anesthesia.

EVALUATION:
Individual scores obtained according to the evaluation table were totaled, the mean score for each test group at each observation time calculated, and the positive sensitization rate calculated from the changes of at least score 1. The sensitizing potential of the test article was evaluated by comparing the group mean scores and the positive rates in each sensitization group and the control group.

Challenge controls:

See details on study design section.

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (MBT)

Positive control results:

In the positive control sensitization group, all animals (5/5) showed erythema and edema of scores 1 to 3 in the site of challenge exposure to 15% MBT solution. The mean score was 2.4 each for 24 and 48 hours after removal of challenge application and the positive rate 100%.
In the site of challenge exposure to liquid paraffin, there were no skin reactions.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5% test article

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5% test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% test article

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5% test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Water (vehicle)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Water (vehicle). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Water (vehicle)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Water (vehicle). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

5% test article

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 5% test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5% test article

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5% test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

15% MBT

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15% MBT. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

15% MBT

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15% MBT. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

15% MBT

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 15% MBT. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

15% MBT

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 15% MBT. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

Liquid paraffin

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: Liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

Liquid paraffin

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormal findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: Liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Skin Sensitization Potential:

Results are shown in Tables 1 and 2 (see attached background material).

In the test article sensitization group, there were no skin reactions in the observation of the site of challenge exposure to the 5% solution of BZ-0145B after challenge exposure, the mean scores at 24 and 48 hours after removal of challenge application both zero, and thus the positive rate 0%. In the area of challenge exposure to water for injection (the vehicle), there were no skin reactions.

In the negative control group, there were no skin reactions in the site of challenge exposure to the 5% solution of BZ-0145B or the 15% solution of MBT.

General Condition:

Results are shown in Table 3 (see attached background material).

There were no abnormalities in the general condition during the observation period in any test group.

Body Weight:

Results are shown in Table 4 (see attached background material).

In the body weight of animals on day 7 after the start of induction, 1/5 animals in the positive control group showed a body weight decrease. In the body weight of the animals on the ending day of observation, 3/10 animals in the test article sensitization group and 3/5 animals in the control group showed a decrease. The degree of decrease was between 2 and 11 g. Its cause was unclear, but it was thought to be caused by stress from the treatment for intracutaneous induction or closed application at the time of challenge exposure, and this it was judged that it was not caused by administration of the test article or the positive control article.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results, the test article (BZ-0145B) did not induce skin sensitization under the conditions of the study.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental Starting Date: 30 January 2014; Experimental Completion Date: 19 February 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

dimethyl sulphoxide

Concentration:

Test item at concentrations of 50%, 25% or 10% w/w in dimethyl sulphoxide.

No. of animals per dose:

Groups of four mice were treated with the test item at each concentration (including vehicle control).

Details on study design:

Test Item Formulation and Experimental Preparation:
For the purpose of the study, the test item was freshly prepared as a solution in dimethyl sulphoxide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

Procedure:
Preliminary Screening Test:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 50% w/w in dimethyl sulphoxide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.

The thickness of each ear was measured, pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test:
Test Item Administration:
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl sulphoxide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Observations:
Clinical Observations:
All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Body Weights:
The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures:
Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

Positive control substance(s):

other: alpha-Hexylcinnamaldehyde, tech., 85%

Positive control results:

A reduced LLNA study was performed to assess the sensitivity of the strain of mouse to a known sensitizer. The reduced LLNA (rLLNA) has been endorsed by the non-Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC).

A group of five animals was treated with 50 µL (25 µL) of α-Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl sulphoxide at a concentration of 25% v/v. A further control group of five animals was treated with dimethyl sulphoxide alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in dimethyl sulphoxide: 25%
Stimulation Index: 4.91
Result: Positive

α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

Parameter:

SI

Remarks on result:

other: Concentration (% w/w) in dimethyl sulphoxide: 10: SI 2.51 25: SI 3.33 50: SI 2.06

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration (% w/w) in dimethyl sulphoxide: Vehicle: 16886.68 dpm 10: 42454.09 dpm 25: 56151.51 dpm 50: 34746.88 dpm

Preliminary Screening Test:

Red colored staining on the ear and fur was noted post dose on Day 1 and at all subsequent observations. Red colored staining on the ears prevented accurate evaluation of erythema on the ears on Days 2 to 6.

No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in dimethyl sulphoxide.

Main Test:

Estimation of the Proliferative Response of Lymph Node Cells:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in the following table:

Concentration (% w/w) in dimethyl sulphoxide	dpm	dmp/Node	Stimulation Index	Result
Vehicle	16886.68	2110.84	na	na
10	42454.09	5306.76	2.51	Negative
25	56151.51	7018.94	3.33	Positive
50	34746.88	4343.36	2.06	Negative

Clinical Observations and Mortality Data:

Red colored staining on the ears and fur was noted in all test animals post dose on Day 1 and at all subsequent observations.

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

Body Weight:

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

other: equivocal

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item producted a positive result for sensitization and was considered to be a sensitizer under the conditions of the test However, it is considered that the result of the study may be regarded as equivocal based on further evaluation of the results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Guinea Pig Maximization Test (GPMT) and LLNA are two in-vivo methods with different approaches to detect potential of skin sensitizers. Both methods are described in the OECD test guidelines (TG) and recognized by regulatory authorities. The following sensitization studies were performed on the test substance (Pigment Red 81:4; BZ-0145B).

Maximization test in Guinea Pigs:

Maximization test in Guinea Pigs (Nomura, 2002) was performed according to the OECD TG 406 (with one irrelevant deviation concerning animal housing temperature) with the test item (BZ-0145B, purity: 100%; solid content 32.6%) suspended in water at concentrations (w/v %):

- 5% (the maximum irritating concentration without necrosis, for intradermal induction).

- 25% (the maximum preparable concentration, for percutaneous induction).

- 5% (the maximum non-irritating concentration, for challenge exposure).

The positive control of Mercaptobenzothiazole (MBT) showed the valid response in guinea pigs.

No reactions in any region of challenge exposure to 5% of test item showed the control group. No skin reactions were detected in animals exposed with the test item (BZ-0145B) in the observation at 24 hours or 48 hours after removal of challenge application.

The test item was identified in this study as a non-sensitizer.

Local Lymph Node Assay in mouse:

The Local Lymph Node Assay in mouse was performed according to the OECD TG 429 (without deviations) with the test item (Pigment Red 81:4, purity: not stated in report) as a solution in dimethyl sulphoxide (DMSO) at concentrations of 50, 25 and 10% w/w.

The used female CBA/Ca (CBA/CaOlaHsd) mouse strain showed the positive response of a known sensitizer α Hexylcinnamaldehyde (tech., 85%). The test item was identified in this study as sensitizer.

Discussion on the Results and Limitations of the Studies:

In the OECD TG 429 is it indicated that in the LLNA are certain limitations that may necessitate the use of OECD TG 406 (GPMT), e.g. false positive findings with certain skin irritants. Pigment Red 81:4 was investigated in the in-vitro EPISKIN reconstructed human epidermis model according to the OECD TG 439 and was found as not irritating. Therefore, the assumption of false-positive skin sensitization potential due to the irritant properties of the test item may be excluded.

In the LLNA report, the test item was identified as skin sensitizer. It should be noted that in the LLNA study DMSO was used as vehicle, whereas in the GPMT study the test item was suspended in water. According to Anderson et al. (2011) DMSO as vehicle augments the LLNA response of certain chemicals. DMSO is a polar solvent and known as a penetration enhancer, wherefore the bioavailability of the allergen across the stratum cornea may be augmented. However, the authors admit vehicle selection has not typically been shown to affect the category of sensitization because the Stimulation Index (SI) value is based on increase in lymphocyte proliferation over vehicle control. Therefore, the assumption of response increase due to DMSO use may be excluded.

In the LLNA report, the middle concentration (25%, w/w) of Pigment Red 81:4 gave a SI of 3.33. In the OECD TG 429 is indicated that the ratio of the mean proliferation (SI) in each treated group to that in the vehicle treated group, which should be ≥ 3 before classification of the test substance as a potential skin sensitizer. Nevertheless, the lower and higher concentrations gave the SI indexes of 2.51 and 2.06, respectively. In ICCVAM (2009) it is stated, that under appropriate test conditions, proliferation of lymphocytes in the lymph node is proportional to the dose applied, and provides a means of obtaining an objective, quantitative measurement of sensitization. Therefore, it may be concluded that there are no dose-response proportionality and the middle concentration exceeding 1.1 times the threshold of the decision may be regarded as equivocal.

According to Basketter and Scholes (1992), LLNA detects chemicals that exhibit a strong sensitization potential in the GPMT. For chemicals classified as moderate sensitizers in the GPMT, LLNA is usually positive or provides an indication of sensitizing activity (that is not sufficient to satisfy the current criteria for regarding the result as positive). Weaker sensitizers in the GPMT are usually not detected by the LLNA. Therefore, it may be assumed that the conclusion of the GPMT study (Nomura, 2002) presents enough of a sensitive result and the non-linear response in the LLNA study may be regarded as equivocal.

Conclusion:

The positive result of LLNA study is not linear and shows no dose-proportionality. Therefore, it may be assumed that the result of GPMT describing non-sensitizing properties of Pigment Red 81:4 is more reliable than the result of LLNA, where the result was equivocal.

Therefore, greater reliance should be placed on the GPMT study result.

Migrated from Short description of key information:

Based on the results of the GPMT study, the test substance does not induce skin sensitization. The results of the LLNA study were equivocal.

Justification for selection of skin sensitisation endpoint:

The GPMT performed with the test item is a reliable study (according to OECD TG 406 and under GLP) and can be used as key study for the test item registration. The LLNA study is performed according to the OECD TG 429 and under GLP; however the interpretation of the result may be contested due to non-conformity with ICCVAM LLNA Performance Standards (2009).

Based on the mentioned reasons, it is considered that the GPMT study is the  key study.

Justification for classification or non-classification

Skin Sensitization:

Based on an evaluation of the available study data, it is concluded that the results of the GPMT are of greater reliability than the results of the LLNA study (equivocal) and that greater reliance shall be placed on the GPMT study.

In the GPMT study the substance was identified as a non-sensitizer as it did not induce skin sensitization under the conditions of the study.

The substance is therefore not classified for skin sensitization.