N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Endpoint summary

Description of key information

- Skin sensitisation: sensitising, male/female, Guinea pig, OECD TG 406, Schneider 1988.

- 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:

09 May 1988 to 09 Jun 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

May 1981

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1984

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 sensitisation for over a decade and is recommended by international test guidelines such as the OECD.

Species:

guinea pig

Strain:

other: Pirbright White Strain (Tif: DHP)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 10 weeks old.
- Weight at study initiation: 340 to 436 g.
- Housing: The animals were housed individually in Macrolon cages (Type 3), assigned to the different groups by means of random numbers generated by the random number generator.
- Diet: standard guinea pig pellets, ad libitum.
- Water: fresh water, ad libitum
- Acclimation period: 1 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 09 May 1988 To: 09 Jun 1988

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

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

Day(s)/duration:

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

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

Injection: 0.1 mL
Treatment: 5.0 % test substance in arachis oil
Control: arachis oil

Day(s)/duration:

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

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

Injection: 0.1 mL
Treatment: 5.0 % test substance in adjuvant/physiological saline mixture, 1:1 (v/v)
Control: adjuvant/physiological saline mixture, 1:1 (v/v)

Day(s)/duration:

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

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

Patch: 2 x 4 cm; approx. 0.4 g per patch
Treatment: 30 % test substance in petrolatum
Control: petrolatum

Day(s)/duration:

Day 8: Occlusive dressing for 48 hours

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

Patch: 2 x 2 cm; approx. 0.2 g paste per patch; occlusive dressing
Both treatment and control: 10 % w/w test substance in petrolatum (one flank), vehicle alone (other flank)

Day(s)/duration:

Day 21: occlusive dressing for 24 hours

No. of animals per dose:

Test group: 10
Control group: 5

Details on study design:

RANGE FINDING TESTS
Intradermal Induction
- Concentrations of test substance and vehicle: 1, 5, 10, and 30 % in petrolatum.

MAIN STUDY
Treatment schedule: A set of three 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 substance and vehicle: 5.0 % in arachis oil.
- Pre-treatment: An area on the neck was shaved prior to the treatment.

Treatment (Day 0): Three pairs of intradermal injections (0.1 mL per injection) were made simultaneously as follows:
(1) adjuvant and physiological saline mixture, 1:1 (v/v)
(2) 5 % test substance in arachis oil
(3) 5 % test compound in the adjuvant physiological saline mixture.

Treatment (Day 8): Epidermal Application Induction
- Pre-treatment: The application sites were pre-treated the day before with 10 % sodium lauryl sulfate (open application).
- Concentration of test substance: 0.2 g paste of 10 % test substance in petrolatum
- Treatment: a filter paper patch was applied to the neck of the animals (patch 2 x 4 cm; occluded administration for 48 hours).


B. CHALLENGE EXPOSURE
- Epidermal Application Challenge (Day 21)
- Vehicle: Same as for epidermal application induction
- Concentration tested, test substance: approx. 0.2 g paste of 10 % (sub-irritant concentration) in petrolatum and the vehicle alone
- Treatment: the animals were tested on the flank with test substance in petrolatum and the vehicle alone (patch 2 x 2 cm; occluded administration for 24 hours). The concentrations of the test substance for the induction and challenge periods were determined on separate animals.
Reactions to challenge were recorded 24 and 48 h after removing the dressings and graded according to Draize scoring scale.

OTHER
- Individual body weights were recorded prior to dosing, on day 1 and at end of the test.

Challenge controls:

No

Positive control substance(s):

yes

Remarks:

2,4-dinitrochlorobenzene (DNCB)

Positive control results:

The reliability check resulted in positive response in 10 (5 males and 5 females of 10 (100%) challenged guinea pigs at 24 and 48 hours after challenge. There were no irritant skin reactions in control groups. Based on results, DNCB was graded as an extreme sensitiser according to Magnusson and Kligman. See also Table 3 in 'Any other information on results incl. tables'.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10 % w/v in petrolatum

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10 % w/v in petrolatum

No. with + reactions:

0

Total no. in group:

20

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:

10 % w/v in petrolatum

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

Scaling recorded in 2 males and 2 females after 24 hrs: very slight to well-defined erythema

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 % w/v in petrolatum

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

Scaling recorded in 4 males and in 5 females after 48 hrs: very slight to well-defined erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1 % DNCB

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Scaling recorded in 5 males and 5 females after 24 hrs: well-defined erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1 % DNCB

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Scaling recorded in 5 males and 5 females after 24 hrs: very slight yo well-defined erythema

Remarks on result:

positive indication of skin sensitisation

RESULTS

Following challenge with a 10 % preparation of test substance in petrolatum, very slight to well-defined erythema was present in 9/20 test and 0/10 control animals, giving a net response of 45 %.

No erythema was present in test or control animals when challenged with vehicle only. Very slight oedema was also present in one test animal at the test site at the 48 h reading. There were no deaths in the test and negative control groups.

Body weights were unaffected by the treatment regimens and all guinea pigs gained weight over the course of the experimental phase.

Table 2 Dermal reactions observed after challenge applications

Scored after		24 hours	48 hours
Test group	10 % test substance	4/20	9/20
	Vehicle only	0/20	0/20
Negative/vehicle control	10 % test substance	0/10	0/10
	Vehicle only	0/10	0/10
Positive control (reference data)	0.1 % DNCB	10/10 (0/10 control)	10/10 (0/10 control)
	Vehicle only	0/10 (0/10 control)	0/10 (0/10 control)

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to the maximisation grading of Magnusson and Kligman the substance showed an extreme grade of skin-sensitising (contact allergenic) potential in albino guinea pigs.

Executive summary:

A dermal sensitisation test was conducted with the test substance using the Maximisation procedure in accordance with OECD TG 406 following GLP principles. Male and female Pirbright White Strain (Tif: DHP) guinea pigs (10 vehicle control, 20 test group and 10 positive control) were included. A topical challenge dose of test substance was administered to both groups. The dose levels for the main study were selected on the basis of screening studies in separate animals to determine the primary irritation threshold concentration. The tested concentrations of 1, 5, 10 and 30% test substance in white petrolatum did not induce erythema reactions. 10% was used as a sub-irritant concentration for the challenge application. The sensitivity of the strain is checked every six months at performing laboratory with paraphenylene-diamine or Potassium-dichromate. In an amendment of the study report, the sensitivity of the strain was confirmed in a March 1988 test with 2,4-dinitrochlorobenzene (DNCB).

The induction was a two-stage operation as follows: at the first induction: 3 pairs of intradermal injections (0.1 mL per injection) were made simultaneously into the shaved neck of the guinea pigs as follows: adjuvant and saline (1:1); test compound 5 % in Oleum arachidis, and test compound test substance 5 % in the adjuvant saline mixture. Second induction entailed: one week later, approx. 0.4 g paste of 30 % test substance was incorporated in petrolatum and applied on a filter paper patch to the neck of the animals (patch 2x4 cm; occluded administration for 48 hours).

Two weeks after the epidermal induction application the animals were tested on the flank with approx. 0.2 g paste of 10 % test substance in petrolatum and the vehicle alone (patch 2x2 cm; occluded administration for 24 hours). Reactions to challenge were recorded 24 and 48 h after removing the dressings and graded according to Draize scoring scale. Individual body weights were recorded prior to dosing, on day 1 and at end of the test.

Following challenge with a 1 0% preparation of test substance in petrolatum, very slight to well-defined erythema was present in 9/20 test and 0/10 control animals, giving a net response of 45 %. No erythema was present in test or control animals when challenged with vehicle only. Very slight oedema was also present in one test animal at the test site at the 48 h reading. There were no deaths in the test and negative control groups. Body weights were unaffected by the treatment regimens and all guinea pigs gained weight over the course of the experimental phase.

Since up to 45 % of the animals were sensitised by the test substance at 10 % under the experimental conditions, the test substance is considered to be a moderate skin sensitiser in the guinea pig.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation, Schneider 1988

A dermal sensitisation test was conducted with the test substance using the Maximisation procedure in accordance with OECD TG 406 following GLP principles. Male and female Pirbright White Strain (Tif: DHP) guinea pigs (10 vehicle control, 20 test group and 10 positive control) were included. A topical challenge dose of test substance was administered to both groups. The dose levels for the main study were selected on the basis of screening studies in separate animals to determine the primary irritation threshold concentration. The tested concentrations of 1, 5, 10 and 30 % test substance in white petrolatum did not induce erythema reactions. 10% was used as a sub-irritant concentration for the challenge application. The sensitivity of the strain is checked every six months at performing laboratory with paraphenylene-diamine or Potassium-dichromate. In an amendment of the study report, the sensitivity of the strain was confirmed in a March 1988 test with 2,4-dinitrochlorobenzene (DNCB).

The induction was a two-stage operation as follows: at the first induction: 3 pairs of intradermal injections (0.1 mL per injection) were made simultaneously into the shaved neck of the guinea pigs as follows: adjuvant and saline (1:1); test compound 5 % in Oleum arachidis, and test compound test substance 5 % in the adjuvant saline mixture. Second induction entailed: one week later, approx. 0.4 g paste of 30 % test substance was incorporated in petrolatum and applied on a filter paper patch to the neck of the animals (patch 2x4 cm; occluded administration for 48 hours).

Two weeks after the epidermal induction application the animals were tested on the flank with approx. 0.2 g paste of 10 % test substance in petrolatum and the vehicle alone (patch 2x2 cm; occluded administration for 24 hours). Reactions to challenge were recorded 24 and 48 h after removing the dressings and graded according to Draize scoring scale. Individual body weights were recorded prior to dosing, on day 1 and at end of the test.

Following challenge with a 10 % preparation of test substance in petrolatum, very slight to well-defined erythema was present in 9/20 test and 0/10 control animals, giving a net response of 45 %. No erythema was present in test or control animals when challenged with vehicle only. Very slight oedema was also present in one test animal at the test site at the 48 h reading. There were no deaths in the test and negative control groups. Body weights were unaffected by the treatment regimens and all guinea pigs gained weight over the course of the experimental phase.

Since up to 45 % of the animals were sensitised by the test substance at 10 % under the experimental conditions, the test substance is considered to be a moderate skin sensitiser in the guinea pig.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

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 substance is classified as 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.