2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Endpoint summary

Description of key information

In vitro data are not available; in vivo data predate the adoption of validated in vitro methods.

The key study for skin sensitisation endpoint is as follows:

Skin sensitisation (OECD 406, GLP, RL1): not sensitising (M-087125-01-1, Stropp, 1997)

The supporting study for skin sensitisation endpoint is as follows:

Skin sensitisation (no guideline, non-GLP, RL2): not sensitisting (M-086893-01-1, Hixson, 1982)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Guinea pig maximization test.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 12 1997 - September 5 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

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was performed prior to adoption of the LLNA (OECD 429)

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Hsd Poc:DH

Sex:

female

Details on test animals and environmental conditions:

Species and Strain:

The animals used were SPF-bred guinea pigs of the strain Hsd Poc:DH supplied by the Harlan Winkelmann GmbH Laboratory Animal Breeders in 33176 Borchen. A total of 32 female animals were used in the study, including those employed in the range-finding test to determine the challenge concentration. In addition, 5 female animals were used to determine the induction concentrations.

This guinea pig strain Hsd Poc:DH has been used for toxicological studies at Bayer AG since years. The state of health of the breeding colony is routinely spot-checked for the main specific pathogens. Appropriate sensitivity of this strain of guinea pig for sensitization tests and appropriate reliability of the experimental technique have been verified and are checked at regular intervals.

Acclimatization: at least seven days

State of Health: Only healthy animals exhibiting no clinical signs were used for the study. The animals were not vaccinated or treated against infections either before receipt, or during the adaptation or study periods. The females were nulliparous and non-pregnant.

Age and Body Weight: The guinea pigs exhibited a mean weight of 299 - 323 grams at the beginning of the study. This weight corresponds to an age of 4 - 5 weeks.


Animal Care:

Animal Accommodation: During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages, in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cages were exchanged for ones with clean bedding two times per week. Low-dust wood shavings supplied by Ssniff Spezialdiaten GmbH, Soest, were used as bedding. The wood shavings were spotchecked for contaminant levels. The analytical results provided no evidence for an effect on the study objective.

Animal Rooms:
All animals used in this study were kept in the same animal room, one in which guinea pigs from other sensitization studies were also housed. Adequate separation, unambiguous cage markings and suitable organization of the work ensured that animals were not mixed up.

Cleaning and Disinfection:
The animal room was swept out with a broom each day, and thoroughly cleaned with water once weekly. The room was disinfected at least once each month with Zephirol® (10 grams benzalkonium chloride per 100 grams disinfectant; working dilution 2%). Contamination of the diet and contact with the experimental animals were avoided during this work. No pest control measures were carried out in the animal rooms. KILLGERM Roach Traps ®, a cockroach trap which uses no pesticides, was placed in the animal room.


Diet: The diet consisted of "Altromin®3020 - Maintenance Diet for Guinea Pigs" supplied by Altromin GmbH in Lage, and of tap water. Food and water were provided ad libitum.
The tap water met drinking water standards.

Climatic Conditions: The room climate in the animal room was set as follows:

Room temperature: 22 ± 3°C (possibly drifting higher at outdoor temperatures above 24°C)

Relative humidity: 40 - 60 %

Light/dark cycle: Twelve hours; artificial lighting from 6 AM to 6 PM

Air exchange rate: >= 10 times per hour

Occasional deviations from these standards occurred at times including the period following the cleaning of the animal room. They had no apparent effect in the course of the study.

Route:

epicutaneous, semiocclusive

Vehicle:

polyethylene glycol

Concentration / amount:

50%

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

2.5%

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

polyethylene glycol

Concentration / amount:

50%

No. of animals per dose:

Dose range finder study: 2
Test item group: 20
Control group: 10

Details on study design:

Intradermal Induction:
The dorsal region and the flanks of the guinea pigs were shorn one day prior to the application. Starting behind the nape of the neck, three injections each in a row were made on the left and the right side of the spinal column. The 1st and 2nd injections were made as contiguous as possible and the 2nd and 3rd injections in a distance of about 2 cm. The volume applied per injection site was 0.1 ml.

The animals of the three groups were treated as follows:

a) Test substance group

1. Injection site: cranial/bilateral, complete Freund's adjuvant diluted 1:1 with sterile physiological saline solution.

2. Injection site: medial/bilateral. 2.5% active ingredient formulated in polyethylene glycol 400.

3. Injection site: caudal/bilateral 2.5% active ingredient formulated at equal parts in sterile physiological saline solution and complete Freund's adjuvant.

b) Control group: The animals of the control group were treated in the same manner as the animals of the test substance group; however, the formulations for injection site pairs 2 and 3 did not contain any test substance but in pair 2 undiluted vehicle and in pair 3 a 50% formulation of the vehicle in a 1:1 mixture FCA/physiological saline solution.


Topical Induction: The topical induction was performed one week after the intradermal induction. On the day prior to topical treatment, the test areas of the animals were shorn. Due to the strong irritation effects at the injection sited of the first induction (up to encrustations), 10% sodium lauryl sulphate was not applied at day 7 for further irritation of the skin. Hypoallergenic patches ( 2 x4 cm) were placed between and on the injection sites, covered with aluminum foil and held securely in place on the skin using a FERMOFLEX self-adhesive tape (TRANSATLANTIC GmbH, Schwarzenbach, Germany).

The patches were treated as follows:

a) Test substance group: 0.5 ml 50% active substance formulated in polyethylene glycol
400

b) Control group: 0.5 ml polyethylene glycol 400, At the end of the 48-hour exposure period, the remaining substance was removed with sterile physiological saline solution.


Grading of the Skin Reaction:
The skin reactions were assessed 48 and 72 hours after the start of the application to induce the challenge and for the range-finding studies to establish concentrations for the topical induction and challenge in accordance with the following pattern:
0 = No reaction
1 = Slight localized redness
2 = Moderate confluent redness
3 = Severe redness and swelling

Descriptions of any dermal findings not covered by this scale were recorded.


Range finding tests:

Dose range-finding study for topical induction: Three different concentrations and the vehicle were tested in each case on four guinea pigs. The patches moistened with 0.5 ml of the test substance formulations were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period, the remaining test substance was removed with physiological
saline solution and twenty-one hours later the treated areas were shorn. The skin reactions were evaluated 48 hours and 72 hours after the start of the application.

Dose range-finding study for challenge: One week prior to the challenge, the challenge concentration was determined on 2 guinea pigs in the main study that were treated in the same manner as the control animals during the inductions. Four patches each loaded with 0.5 ml test substance formulation or the vehicle were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period, the remaining test substance was removed with physiological saline solution and twenty-one hours later the treated areas were shorn. The skin
reactions were evaluated 48 hours and 72 hours after the start of the application.

Challenge controls:

The challenge was performed three weeks after the intradermal induction. In the meantime, the test substance concentrations for the challenge had been determined in a dose range-finding study using 2 guinea pigs that were treated during the inductions in the same manner as the control animals.

The dorsal region and the left flank of the animals were shorn one day prior to the challenge. During the challenge a hypoallergenic patch loaded with a 50% test substance formulation was placed on the left flank (caudal) of the animals of the test substance group and the control group and held securely in place on the skin with a FERMOFLEX self-adhesive tape for 24 hours. A patch loaded only with the vehicle was placed also on the left flank (cranial) as control. The volume applied in each case was 0.5 ml. At the end of the exposure period, the remaining test substance was removed with physiological saline solution, and twenty-one hours later the skin of the animals was shorn in the zone of the challenge area.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Positive control results:

60% positive reaction

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

intradermal induction: 0%
topical induction: 0%
challenge: 50%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

intradermal induction: 0%
topical induction: 0%
challenge: 50%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

intradermal induction: 2.5%
epicutaneous induction: 50%
Challenge: 50%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight localized redness in one animal

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

intradermal induction: 2.5%
epicutaneous induction: 50%
Challenge: 50%

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

1st reading

Group:

positive control

Dose level:

intradermal induction: 2.5%
topical induction: 40%
challenge: 40%

No. with + reactions:

6

Total no. in group:

10

Appearance and behaviour of the test substance group were not different from the control group. At the end of the study the body weight in the test substance group was slightly higher than in the control group. The animals in the test substance group and in the control group showed reddening and wheals at the injection sites after 24 hours. Reddening, wheals and encrustations at the injection sites of the first induction were recorded after 2 and 7 days with the most severe effects after 7 days.

Interpretation of results:

GHS criteria not met

Conclusions:

In a Guinea Pig Maximization Test, Triflumuron was examined in female guinea pigs for its skin sensitizing properties. The challenge using a 50% test substance formulation led to slight skin effects (grade 1) in 1/20 animals in the treatment group after 48 hours and in none of the animals in the treatment group after 72 hours. In the control group none of the animals showed skin effects after 48 and 72 hours.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results of the Maximisation assay with triflumuron demonstrated no skin sensitisation potential. Therefore, triflumuron does not meet the criteria for classification for skin sensitisation according to Regulation (EC) No. 1272/2008.