1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Endpoint summary

Description of key information

- Skin sensitisation: sensitising, male/female, Guinea pig, OECD TG 406, Sommer 1999

- Respiratory sensitisation: not expected to be of concern for respiratory sensitisation

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Aug 1999 to 26 Aug 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

July 17, 1992

Qualifier:

according to guideline

Guideline:

other: 96/54/EC, IV.C

Version / remarks:

July 30, 1996

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Currently no LLNA study is available for assessment. The GPMT test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.

Species:

guinea pig

Strain:

other: Himalayan Spotted [GOHI Ibm: GOHI (SPF)]

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age/weight at dosing: Approximately 1-2 months / 320-431 g
- Housing: Individually in Macrolon Type 3 cages
- Acclimatisation period: At least 5 days
- Diet: Ad libitum
- Water: Municipal water supply ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 50 ± 20%
- Air changes: Approximately 13 - 14 per hour
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES: 2 Aug 1999 to 26 Aug 1999

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

Injection: 0.1 mL
Treatment: adjuvant/physiological saline mixture, 1:1 (v/v)
Control: adjuvant/physiological saline mixture, 1:1 (v/v)

Day(s)/duration:

Day 0: First injection out of the three pairs of intradermal injections

Route:

intradermal

Vehicle:

other: peanut oil

Concentration / amount:

Injection: 0.1 mL
Treatment: 5% in peanut oil
Control: peanut oil

Day(s)/duration:

Day 0: Second injection out of the three pairs of intradermal injections

Adequacy of induction:

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

Route:

intradermal

Vehicle:

other: peanut oil

Concentration / amount:

Injection: 0.1 mL
Treatment: 5% test substance in adjuvant/physiological saline mixture, 1:1 (v/v)
Control: peanut oil, 50% (w/v) with adjuvant/physiological saline mixture, 1:1 (v/v)

Day(s)/duration:

Day 0: Third injection out of the three pairs of intradermal injections

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

Patch: 2x4 cm; approx. 0.4 g per patch; occlusive dressing
Treatment: 100% test substance
Control: vaseline

Day(s)/duration:

Day 8: Occlusive dressing for 48 hours

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

Concentration: 30% test substance (both control as treatment group)
Amount: 0.35 mL
Two treatments were placed on all animals: test substance on the test flank and vehicle on the vehicle flan

Day(s)/duration:

Day 21: occlusive dressing for 48 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 animals in the vehicle control group and 20 animals in the test group

Details on study design:

RANGE FINDING TESTS:
Intravenous Inderuction Pretest
- Concentrations of test item and vehicle: 0.5, 1.0, 3.0 and 5.0% in peanut oil
- Pre-treatment: A 5-cm wide area in the neck-shoulder region of 1 male and 1 female animal was shaved approximately 1 hour before treatment.
- Treatment: One pair of intradermal injections (0.1 mL) of a 1:1 mixture of FCA/physiological saline and one pair of injections for each concentration were administered, one of each pair on each side of the spine.
- Observations: The test sites were examined 24 and 48 hours after administration to determine the highest concentration to produce mild to moderate irritation without systemic toxicity.
 
Epidermal Induction Pretest
- Concentrations of test item and vehicle: 30, 50 and 80% in vaseline and 100% (undiluted).
- Pretreatment: Two pairs of intradermal injections (0.1 mL) of a 1:1 mixture of FCA/physiological saline were administered to 1 male and 1 female animal.
- Treatment: Seven days later, the test item/vehicle mixtures and the undiluted test item were applied with four Hill Top Chambers, two on each flank of both animals.
- Observations: The test sites were examined 24 and 48 hours after administration to determine the highest concentration to cause mild to moderate irritation for the induction application and no irritation for the challenge application (highest non-irritant dose).

MAIN STUDY:
Treatment schedule: A set of intradermal induction injections was made on Day 0. An epidermal induction application was made once on Day 8. The epidermal challenge application was made once on Day 21.

A. INDUCTION EXPOSURE
- Intradermal Induction Injections: Concentration of test item and vehicle: 5.0% in peanut oil.
- Pretreatment: An area approximately 5 cm x 5 cm on the back of the neck was shaved approximately 1 hour before treatment.
 
Treatment (Day 0): Three pairs of injections (0.1 mL in volume) were applied into the shaved area by making sure that one of each pair was on each side of the midline.
- Vehicle control group injections:
(1) adjuvant/physiological saline mixture, 1:1 (v/v)
(2) peanut oil
(3) peanut oil, 50% (w/v) with 1:1 adjuvant/physiological saline mixture
- Test item group injections:
(1) adjuvant/physiological saline mixture, 1:1 (v/v)
(2) test substance in peanut oil
(3) test substance in 1:1 adjuvant/physiological saline mixture
 
Treatment (Day 8): Epidermal Application Induction
- Concentration of test item: 100% (undiluted test item)
- Treatment: A filter paper patch was fully loaded (approximately 0.4 g) with the test Item (test item group) or vaseline vehicle alone (vehicle control group) and held in place with the occlusive dressing for 48 hours.
 
B. CHALLENGE EXPOSURE
- Epidermal Application Challenge (Day 21)
- Vehicle: Vaseline
- Concentration tested, test item: 30% (highest non-irritant dose)
- Pretreatment: The flanks of all animals were shaved immediately prior to treatment.

Treatment (Day 21): One chamber loaded with the test item/vehicle mixture (approximately 0.35 mL) was placed on one flank (test flank) and one chamber loaded with the vehicle alone was placed on the other flank (vehicle flank) of the animals of both groups. The chambers were held in place with the occlusive dressing for 24 hours.
- Period of observation: Through 48 hours after completion of the challenge application.
- Skin irritation check: Application sites were examined 1 hour after removal of the epidermal induction dressing on Day 10 and rated for positive or negative skin irritation reactions.
- Scoring intervals: 24 and 48 hours after removal of the dressing for the challenge application.
- Grading of skin reactions: Dermal reactions were graded according to the Draize scale

OTHER:
- Method of sacrifice: CO2 asphyxiation
- Clinical observations: Checked daily; any remarkable findings, with indication of severity, were recorded.
- Mortality: Checked daily
- Body weight: Measured and recorded immediately before treatment and at study termination.
- Grading of skin reactions: Please, see 'Any other information on materials and methods incl. tables' section

Challenge controls:

Vehicle without test substance

Positive control substance(s):

yes

Remarks:

Benzocaine 30%, checked periodically

Positive control results:

Latest Reliability Check: Very slight or well defined erythema was seen in 12/20 test animals, 24 hours after challenge, and in 10/20 test animals 48 hours after challenge. There was no erythema seen in any of the controls. The net response was, therefore, 60% and benzocaine was classified as a moderate skin sensitiser and confirmed the sensitivity of the test system.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

30%

No. with + reactions:

6

Total no. in group:

20

Clinical observations:

no clinical signs observed

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

30%

No. with + reactions:

10

Total no. in group:

20

Clinical observations:

no clinical signs observed

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

30% vaseline

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no clinical signs observed

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

30% vaseline

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no clinical signs observed

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

30 % benzocaine

No. with + reactions:

12

Total no. in group:

20

Clinical observations:

Very slight or well defined erythema

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

30 % benzocaine

No. with + reactions:

10

Total no. in group:

20

Clinical observations:

Very slight or well defined erythema

Remarks on result:

positive indication of skin sensitisation

Table 1. Magnusson and Kligman test: Number of animals with positive signs of allergic skin reactions following challenge

	Challenge with 30% test substance		Challenge with Vaseline
Scored after:	24 hours	48 hours	24 hours	48 hours
Main test – test group	6/20	10/20	0/20	0/20
Main test – negative vehicle control	0/10	0/10	0/10	0/10
	Challenge with 30% benzocaine
	24 hours		48 hours
Positive control – test group	12/20		10/20
Positive control – vehicle control	0/10		0/10

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In a dermal sensitisation study performed in compliance with GLP and following the OECD 406 guideline, positive skin reactions on the test flanks were observed in 2 males and 4 females of the test group animals at the 24-hour examination and in 5 males and 5 females at the 48-hour examination; the sensitisation rate for the test substance was therefore 50%. Desquamation was seen in 3 males and 3 females at the 48-hour observation. Therefore, the test substance is considered to be a skin sensitiser.

Executive summary:

In a dermal sensitisation study performed in compliance with GLP and following the OECD 406 guideline, young adult, male and female, Himalayan Spotted (GOHI Ibm:GOHI (SPF) guinea pigs (10/sex test group, 5/sex vehicle controls) were tested with the test substance using the method of Magnusson and Kligman. For the main study, the concentrations used were: intradermal injections 5.0% test substance in peanut oil; epidermal induction application undiluted test substance; epidermal challenge applications 30% test substance in Vaseline and vehicle alone. From the findings of a pilot study, 30% test substance concentration was the highest concentration that caused no skin irritation.

A positive control experiment with benzocaine was performed and confirmed the sensitivity of the test system. Positive skin reactions on the test flanks were observed in 2 males and 4 females of the test group animals at the 24-hour examination and in 5 males and 5 females at the 48-hour examination; the sensitisation rate for the test substance was therefore 50%. Desquamation was seen in 3 males and 3 females at the 48-hour observation. There were no allergic skin reactions on the vehicle flanks and in the vehicle control group. There was no mortality, and there were no remarkable clinical observations in either group. Body weights were not affected by treatment. Therefore, the test substance is considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

All available data was assessed and the studies representing the worst-case effects were included as key studies. All other information was included as supporting information. The in vitro data requirements were waived based on the availability of adequate in vivo data. The test substance is considered to be a skin sensitiser based on the results of the key study. 

Skin Sensitisation

In the key study (Sommer 1999), performed in compliance with GLP and following the OECD TG 406, young adult, male and female, Himalayan Spotted (GOHI Ibm:GOHI (SPF) guinea pigs (10/sex test group, 5/sex vehicle controls) were tested with the test substance using the method of Magnusson and Kligman. For the main study, the concentrations used were: intradermal injections 5.0% test substance in peanut oil; epidermal induction application undiluted test substance; epidermal challenge applications 30% test substance in Vaseline and vehicle alone. From the findings of a pilot study, 30% test substance concentration was the highest concentration that caused no skin irritation.

A positive control experiment with benzocaine was performed and confirmed the sensitivity of the test system. Positive skin reactions on the test flanks were observed in 2 males and 4 females of the test group animals at the 24-hour examination and in 5 males and 5 females at the 48-hour examination; the sensitisation rate for the test substance was therefore 50%. Desquamation was seen in 3 males and 3 females at the 48-hour observation. There were no allergic skin reactions on the vehicle flanks and in the vehicle control group. There was no mortality, and there were no remarkable clinical observations in either group. Body weights were not affected by treatment. Therefore, the test substance is considered to be a skin sensitiser.

A supporting study (Ullmann 1978) is available. The study was performed according to an AFDO (1959) guideline and was not GLP compliant. The test substance was administered to male and female guinea pigs as an intradermal injection and epicutaneous application at a dose of 0.1 mL 0.1 % dilution in the vehicle. No differences between the test group and the vehicle-treated controls were seen, after either intradermal or epidermal challenge application of the test substance. The test substance was found to be devoid of skin sensitising (contact allergenic) potential in albino guinea pigs.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The test substance is not expected to be of concern for respiratory sensitisation. Following the recommended approach outlined in R.7.3.12.3, the test substance is not expected to be a respiratory sensitiser because the test substance is not di-isocyanate or protein. Furthermore, no structural warnings for respiratory sensitisation were found using the QSAR toolbox (v4.4).

Justification for classification or non-classification

Based on the available information, the test substance is classified as a skin sensitiser category 1, H317: May cause an allergic skin reaction in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.