(1R,2S,5S)-2-(methoxycarbonyl)-6,6-dimethyl-3-azoniabicyclo[3.1.0]hexane chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Buehler Test

Type of information:

experimental study

Remarks:

Buehler Test

Adequacy of study:

key study

Study period:

28 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The method was designed to meet the OECD Guideline for Testing of Chemicals: Skin sensitisation study (TG 406, adopted 2021)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

2021

Deviations:

yes

Remarks:

A part of time points of temperature deviated from the study plan, the smallest and biggest deviated values were 23.1℃ and 26.0℃, respectively. so these deviations were considered not to affect the quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

Guinea pig is the preferred strain for the heredity characters, stability and available background data. Based on the Skin Sensitisation result of positive control test item α-Hexylcinnamaldehyde, this strain is reliable.

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2S,5S)-2-(methoxycarbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-ium chloride

EC Number:

611-400-5

Cas Number:

565456-77-1

Molecular formula:

C9H15NO2HCl

IUPAC Name:

(1R,2S,5S)-2-(methoxycarbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-ium chloride

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Supplier Shenyang Tenghua Biotechnilogy Co., Ltd
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Microbiological status of animals, when known: Unknown
- Age at study initiation:42-56 days on arrival, in the range of 51-65 days at the commencement of first induction.
- Weight at study initiation: Mean weight of treated group 272g ±22 (standard deviation)
- Housing: Animals were housed in the animal room of Building A115. The plastic cage size was
L80 cm×W50 cm×H20 cm. Each cage held no more than 12 animals and animals were identified with a cage card bearing cage number, animal number, study number, group number and SD’s name. Animals used for preliminary irritation test were housed in a separate cage after grouping. Qualified corn cob bedding was used during the test
- Diet (e.g. ad libitum): Diet was available to the animals ad libitum during test
- Water (e.g. ad libitum): Water was available to the animals ad libitum during test
- Acclimation period: Animals were acclimatized to the laboratory for 9 days prior to the test
- Indication of any skin lesions: No
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature and humidity were controlled automatically and recorded daily. The temperature values in the animal room were 20～26℃ (target value 20～23℃).
- Humidity (%): The relative humidity values were 48～70% (target value 40%～70%).
- Air changes (per hr): unknown
- Photoperiod (hrs dark / hrs light): The lighting sequence was 12 hours light, 12 hours dark.
- IN-LIFE DATES: From: To: unknown

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

Purified water

Concentration / amount:

neat test item

Day(s)/duration:

Day 0 (first induction date), Day 7 and Day 14 for 6 hours.

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

purified water

Concentration / amount:

0.2g of neat test material

Day(s)/duration:

Day 28 for 6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

2 for preliminary test, 10 in control group, 20 in treated group

Details on study design:

RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6hrs
- Test groups: Once each week for three weeks (On Day 0 (first induction date), Day 7 and Day 14), about 0.2 g test item (moistened with 0.1 mL purified water) on a piece of filter paper
(about 2 cm×2 cm) to the left side of clipped area of the animal, and placed a piece of
patch (a gauze which was stuck to one layer non-allergenic medical adhesive tape). And
then the patch was wrapped with the non-allergenic medical adhesive tape for 6 hours.
- Control group: The guinea pigs were maintained under identical environmental conditions and were not be treated with the test item. Only at challenge, the guinea pigs were treated with the test item.
- Site: left flank
- Frequency of applications: Day 0 (first induction date), Day 7 and Day 14
- Duration: Day 0 (first induction date), Day 7 and Day 14 for 6 hours
- Concentrations: 0.2g neat test material

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28
- Exposure period: 6hrs
- Test groups: On day 28, the test item was applied to the right site of the depilated back of each animal in both treated and control groups. The operation procedure was the same as that in the
induction phase
- Control group: see above
- Site: right flank
- Concentrations: 0.2g of neat test material
- Evaluation (hr after challenge): 24 and 48 hours after removing the patch, the Guinea
pigs were observed and scored for erythema and edema

OTHER:

Challenge controls:

On day 28, the test item was applied to the right site of the depilated back of each animal
in both treated and control groups. The operation procedure was the same as that in the
induction phase.

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde

Results and discussion

Positive control results:

see table 5 below

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.2g of neat material only treated in challenge phase

No. with + reactions:

0

Total no. in group:

10

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.2g neat material treated only during challange

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10%

No. with + reactions:

6

Total no. in group:

20

Results 4

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

10%

No. with + reactions:

6

Total no. in group:

20

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.2 g of neat test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No visible changes in skin reactions (erythema and/or swelling) were found on the
treated skins during the first, second and third inductions at 48 h observations after the patches were removed and the scores were all 0.

Remarks on result:

no indication of skin sensitisation

Results 6

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.2 g of neat test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No visible changes in skin reactions (erythema and/or swelling) were found on the
treated skins

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Table 4. Skin sensitisation incidence and severity index

Group	Time (h)	Reaction Score				Severity Index		Incidence
		0	1	2	3	24 h	48 h
Control	24	10	0	0	0	0	0	0/10 (0%)
	48	10	0	0	0
Treated	24	20	0	0	0	0	0	0/20 (0%)
	48	20	0	0	0

Table 5. Historical data-skin sensitisation result of positive control

α-Hexylcinnamaldehyde（Study No:G2155E0010）

Group	Time (h)	Score				Severity index		Incidence
		0	1	2	3	24 h	48 h
Control	24	10	0	0	0	0.0	0.0	0/10 (0%)
	48	10	0	0	0
Treated	24	14	5	1	0	0.35	0.35	6/20 (30%)
	48	14	5	1	0

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on above results, PF-04349713-01 is not a skin sensitizer to Guinea pigs. The
results do not meet the GHS classification criteria for skin sensitisation (Buehler Test).

Executive summary:

Induction Phase: No visible changes in skin reactions (erythema and/or swelling) were found on the treated skins during the first, second and third inductions at 24 h and 48 h observations after the patches were removed and the scores were al.

Challenge Phase: No visible changes in skin reactions (erythema and/or swelling) were found on the right treated skin area of each animal in the control and treated animals observed at 24 h and 48 h observations after the patches were removed and the scores were all 0. The incidences of skin sensitisation and severity indexes were all 0% at 24 h and at 48 h after removal of the test item patches.