pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Endpoint summary

Description of key information

- Skin sensitisation: negative, Guinea pig maximization test, OECD 406, Winkler 1997

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:

21 Apr 1997 to 15 may 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Currently no LLNA study is available for assessment. The Guinea pig maximisation 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) GP 43

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: not specified
- Weight at study initiation: 365 to 436 g (for males), 333 to 407 g (for females)
- Housing: The animals were housed individually in Macrolon cages (Type 3)
- Diet: standard guinea pig pellets ad libitum
- Water: fresh water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12

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 of the three pairs of intradermal injections

Route:

intradermal

Vehicle:

peanut oil

Concentration / amount:

Injection: 0.1 mL
Treatment: 3.0% test substance in peanut oil
Control: peanut oil

Day(s)/duration:

Day 0: Second injection 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: physiological saline and peanut oil

Concentration / amount:

Injection: 0.1 mL
Treatment: 3.0% test substance in the adjuvant/physiological saline mixture (w/v) and peanut oil (50%; w/v)
3.0% CGA 215944 tech. in
Control: adjuvant/physiological saline mixture (w/v) and peanut oil (50%; w/v)

Day(s)/duration:

Day 0: Third injection 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:

other: Vaseline

Concentration / amount:

Patch: 2 x 4 cm; approx. 0.4 g per patch; occlusive dressing
Treatment: 50% test substance in vaseline
Control: vaseline

Day(s)/duration:

Day 8: occlusive dressing for 48 hours

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Vaseline

Concentration / amount:

Patch: Hilltop chamber; approx. 0.35 ml per chamber; occlusive dressing
Both treatment and control: 30% test substance in vaseline (one flank), vaseline (other flank)

Day(s)/duration:

Day 22: occlusive dressing for 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Treatment: 10
Control: 5

Details on study design:

RANGE FINDING TESTS:
Intradermal Induction
The vehicle, peanut oil, was selected with respect to the suspendability of the test article and lack of sensitizing properties. The concentration for intradermal injections was selected on account of the solubility of the test article in standard vehicles, the local irritant potency and the systemic tolerability of the test article in a pre-test. The following concentrations of the test substance were examined in pre-test animals in duplicate: 0.5, 1, 3 and 5% (w/v) in peanut oil. Local reactions at the injection sites were examined 24 and 48 hours after duplicate intradermal injections.

Epidermal Applications (induction and challenge)
The concentration for the epidermal applications was selected on account of the primary irritation potential of the test article. The following concentrations were examined in pre-test animals in duplicate to determine the maximum sub-irritant concentration: 10, 30 and 50% (w/v) in vaseline. At least 7 days before application of the test substance, two pairs of consecutive intradermal injections of an adjuvant/saline mixture 1:1 (v/v; 0.1 ml per injection) were applied to the shaved neck of 2 guinea pigs. On each animal, 3 different concentrations were tested simultaneously on the left and right flank using Hilltop chambers. Accordingly, each concentration was tested in duplicate. Naive skin site served as control (not reported). Occlusive dressing was applied for 24 hours. Skin responses were scored 24 and 48 hours after removal of the dressing according to the Draize scale.

MAIN STUDY
A. INDUCTION EXPOSURE (day 0)
Three pairs of intradermal injections (0.1 mL) were made consecutively on the left and right side of the shaved neck in test group, control group and positive control group animals as follows:
- Test groups: #1 adjuvant/physiological saline mixture 1:1 (v/v); #2 3.0% test substance in peanut oil; #3 3.0% test substance in the adjuvant/physiological saline mixture (w/v) and peanut oil (50%; w/v)
- Control group: Injections: #1 adjuvant/physiological saline mixture 1:1 (v/v); #2 peanut oil; #3 adjuvant/physiological saline mixture (w/v) and peanut oil (50%; w/v)
- Positive control group (historical data): 1% mercaptobenzothiazole in peanut oil.

B. EPIDERMAL INDUCTION APPLICATION (day 8):
- In the test group, a 50% solution of the test substance was applied on a filter paper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g per patch and held in place under an occlusive dressing for 48 hours).
- The control group was treated the same way, but with the vehicle (Vaseline) only.
- Positive control group (historical data): 50% mercaptobenzothiazole in Vaseline.
- Irritation was assessed on day 10, 1 hour after removal of the dressings.

C. CHALLENGE EXPOSURE (day 22):
- After 2 weeks without treatment, the test and negative control groups were challenged on one flank with a 30% solution of the test substance in Vaseline and on the other flank with the vehicle (Vaseline) alone using Hilltop chambers (approx. 0.35 ml volume per chamber; occlusive dressing for 24 hours).
- Positive control group (historical data): 10% mercaptobenzothiazole in Vaseline.
- Evaluations: The challenge reactions were assessed 24 and 48 hours after removing the dressings, according to the Draize scale.

OTHER:
Body weights were recorded at start and on conclusion of the test and tabulated with means and standard deviations.

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (measured twice a year)

Positive control results:

Epidermal challenge of 10 male and 10 female guinea pigs with mercaptobenzothiazole (positive control group) resulted in positive responses in all guinea pigs after 24 and 48 hours, corresponding to a sensitization rate of 100%. Scaling was recorded in 7 males (no. 66, 67, 69, 70, 71, 74 and 75) and in all females after 48 hours. No irritant skin reactions were recorded for control animals.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0% w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0% w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

30% w/v

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

None

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

30% w/v

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

other: negative control for pre-test

Dose level:

0% w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

other: negative control for pre-test

Dose level:

0% w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

None

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Scaling was recorded in 7 males and in all females after 48 hours.

Remarks on result:

positive indication of skin sensitisation

Intradermal Induction Pre-test

After duplicate testing of the test substance at 0.5, 1, 3 and 5% concentration in peanut oil, irritation was seen at the site of intradermal injection with all concentrations. Concentration of 3.0% was suitable for injection, systemically well tolerated and chosen for intradermal induction in the maximization test owing to mild to moderate skin irritating properties.

 

Epidermal Application Pre-test

Incorporation of the test article into vaseline was limited to 50%. After duplicate testing at 10, 30 and 50% concentration, irritant skin reactions were seen in animals treated with 50% of the test article. The concentration of 30% the test substance was the highest non-irritant dose. A concentration of 50% was selected for epidermal induction and at 30% for the topical challenge.

 

Guinea Pig Maximization Test

After removal of the occlusive dressing on day 10 after epidermal induction, irritation at the application site was seen in all 20 guinea pigs of the test groups.

Epidermal challenge of 10 male and 10 female guinea pigs (test groups) resulted in a positive response in 1 male guinea pig after 24 hours, and in no positive responses after 48 hours, corresponding to a sensitization rate of 5%. No irritant skin reactions were recorded for control animals.

All animals survived to the scheduled sacrifice. Body weights were not affected by the treatment. The test substance was graded as a weak sensitizer, the lowest grade of the Magnusson and Kligman grading scale.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is graded as a weak sensitiser according to Magnusson and Kligman, which is the lowest possible grade however based on the sensitisation rate of 5%, which is below the threshold of significance (i.e. below 30% according to CLP)

Executive summary:

In a dermal sensitisation study male and female Himalayan spotted (GOHI) Guinea pig (10/sex), were tested using the guinea pig maximization test (adjuvant test) according to Magnusson and Kligman (1980) and OECD 406. The study was performed under GLP conditions. Two main procedures were involved in the study; (a) the potential induction of an immune response; (b) a challenge of that response. From the findings of a pilot study the concentrations for use in the main study were selected. On the basis of the results, a 3% w/v preparation of the test substance in physiological saline was used for intradermal induction, a 50% w/v preparation in Vaseline was used for the epidermal induction and a 30% w/v preparation in Vaseline was used for the challenge. Epidermal challenge of 10 male and 10 female guinea pigs with the test substance resulted in positive response in 1 male guinea pig after 24 hours, corresponding to a sensitisation rate of 5%. No irritant skin reactions were recorded among control animals. Epidermal challenge test of 10 male and 10 female guinea pigs with 1% mercaptobenzothiazole (positive control groups, performed twice yearly) resulted in positive responses in all animals after 24 and 48 hours, corresponding to a sensitisation rate of 100%. The test substance was graded as a weak sensitizer according to the Magnusson and Kligman maximisation scale. Based on the sensitisation rate of 5%, which is below the threshold of significance (i.e. below 30%), classification of the test substance as to its sensitising properties is not required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

All available data was assessed and the studies representing the worst-case effects were included as key studies. The other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

Skin sensitisation (GPMT)

In a dermal sensitisation study male and female Himalayan spotted (GOHI) Guinea pig (10/sex), were tested using the guinea pig maximization test (adjuvant test) according to Magnusson and Kligman (1980) and OECD 406 (Winkler, 1997). The study was performed under GLP conditions. Two main procedures were involved in the study; (a) the potential induction of an immune response; (b) a challenge of that response. From the findings of a pilot study the concentrations for use in the main study were selected. On the basis of the results, a 3% w/v preparation of the test substance in physiological saline was used for intradermal induction, a 50% w/v preparation in Vaseline was used for the epidermal induction and a 30% w/v preparation in Vaseline was used for the challenge. Epidermal challenge of 10 male and 10 female guinea pigs with the test substance resulted in positive response in 1 male guinea pig after 24 hours, corresponding to a sensitisation rate of 5%. No irritant skin reactions were recorded among control animals. Epidermal challenge test of 10 male and 10 female guinea pigs with 1% mercaptobenzothiazole (positive control groups, performed twice yearly) resulted in positive responses in all animals after 24 and 48 hours, corresponding to a sensitisation rate of 100%. The test substance was graded as a weak sensitizer according to the Magnusson and Kligman maximisation scale. Based on the sensitisation rate of 5%, which is below the threshold of significance (i.e. below 30%), classification of the test substance as to its sensitising properties is not required.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data on skin sensitisation, classification is not warranted according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.