Benzamide, N-(4-methyl-3-nitrophenyl)-4-[(4-methyl-1-piperazinyl)methyl]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3. february 2003 - 8. april 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

LLNA method was not available yet at the time the study was conducted.

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-Methyl-3-nitro-phenyl)-4-(4-methyl-piperazin-1-ylmethyl)-benzamide

EC Number:

611-622-2

Cas Number:

581076-60-0

Molecular formula:

C20H24N4O3

IUPAC Name:

N-(4-Methyl-3-nitro-phenyl)-4-(4-methyl-piperazin-1-ylmethyl)-benzamide

Constituent 2

Reference substance name:

N-(4-Methyl-3-nitro-phenyl)-4-(4-methyl-piperazin-1-ylmethyl)-benzamid roh

IUPAC Name:

N-(4-Methyl-3-nitro-phenyl)-4-(4-methyl-piperazin-1-ylmethyl)-benzamid roh

Details on test material:

Batch No.: 022401
Appearance: yellowish powder with lumps
expiration date: december 2003
purity: > 99%
stability at conditions of storage: stable

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Female healthy young adult and non pregnant animals.
Age of the animals: Approx. 5 - 7 weeks at the fist application
Weight range of the animals: 289 g to 336 g at the fist application.
Number of the animals in the main study: 10 animals for the test substance froup and 5 animals for the control group.
Hygiene: optimal hygienic conditions
Room temperature: average of 21.9 C
Relative humidity: average of 48.6%
Air exchange: Approx 12H
Light: Only artificial light from 6.00 a.m. to 6.00 p.m.
Cages: Until Day 0: Makrolon cages type Ill (23 cm x 39 cm bottom area, 18 cm height) with wire m
esh lids, single caging.
From Day O: group cagirg in plastic containers (46 cm x 105 cm x 36 cm), partly shaded, 6 (control
group) or 11 (test substance group) animals per contcliner.
Feed: Altromin Maintenance Diet No. 3122, rich in crude fiber, ad libitum, offered in stainless steel con
tainers. Analysis of the feed for ingredients and contclminants are performed randomly by
Altromin GmbH, D-32791 Lage.
Bedding material: Wood chips (aspen) from Fa. ABEDD Dominik Mayr KEG,
A-8580 Koflach. Reduction of microorganisms by autoclaving.
Water: Tap water offered in Makrolon bottles with stainless steel canules ad libitum.
Identification of the animals: Numbers tattooed in the pinna of the right ear.
Acclimatisation: 5 days.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

about 0.5 g of test substance or about 0.5 g of white petrolatum were applied to each animal

Day(s)/duration:

2

Induction 2

Route:

intradermal

Vehicle:

DMSO

Concentration / amount:

final concentration of the test substance: 2% (w/v) in 3% DMSO in corn oil (v/v), 1+1 (v/v) blended with FCA

Day(s)/duration:

0-1

Induction 3

Route:

intradermal

Vehicle:

DMSO

Concentration / amount:

test substance, 2% (w/v) in 3% DMSO in corn oil (v/v)
a volume of 0.1 ml was applied for each injection site

Day(s)/duration:

0-1

Induction 4

Route:

intradermal

Vehicle:

physiological saline

Remarks:

Injection row: Cranial. Freund's complete adjuvant (FCA), 1+1 (v/v) blended with physiological saline

Concentration / amount:

a volume of 0.1 ml was applied for each injection site.

Day(s)/duration:

0 - 1

Challenge

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

About 0,5 g of test substance formulation or about 0,5 g of white petrolatum were applied to each animal.

Day(s)/duration:

24

Adequacy of challenge:

not specified

No. of animals per dose:

10 of animals in test group
5 of animals in control grou

Details on study design:

According to the Guidelines, the concentrations of the test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to moderate skin irritation. The concentration used for the challenge exposure should be the highest non irritant one.
To obtain the appropriate concentrations of the test substance for the definitive study, a preliminary
test was carried out with 3 female guinea pigs. FCA was administered intradermally and 8 days later 4 concentration of the test substance were administered epicutaneously. The modes of application were the same as in the definitive study. The duration of the epicutaneous exposure was 24 hours. The test substance was dissolved in DMSO: corn oil (v/v) for the intradermal injections and was incorporated in white petrolatum (w/v) for the epicutaneous administration.
For the main study the following concentrations of "STl571 F5" were selected:
2% 8w/v) in 3% DMSO in corm oil (v/v) for the intradermal induction, 25% 8w/w) in white petrolatum for the epicutaneous induction and 10% (w/w) in white petrolatum for the challenge exposure.
Day 0 was 11 February 2003.
Day 0: removal of hair, recording of body weight, intradermal induction exposure
Day 1: examination of injection sites
Day 7: removal of hair, epicutaneous induction exposure
Day 9: end of the epicutaneous induction exposure.
Day 10: skin examination.
Day 21: removal of hair, epicutaneous challenge exposure
Day 22: end of the epicutaneous challenge period.
Day 23: approximately 21 hours after removing the patch cleaning of the challenge area,
approximately 3 hours later skin examination.
Day 24: skin examination, recording of body weight, sacrifice of animals, end of test.
All animals were observed once daily for behavioural changes or signs of toxicity.
The body weight of each animal was recorded on Days 0 and 24.
The application sites were examined 24 hours after the intradermal induction, 24 hours after the end of epicutaneous induction exposure and 24 and 48 hours after the end of the epicutaneous challenge exposure (blind reading of test and control animals).
A skin reaction after the challenge exposure was regarded as positive when the site, where test substance formulation was applied, was more irritated than the area of the site, where the vehicle was
applied. The rate of these positively reacting animals in the test substance group minus the rate
of positively reacting animals in the negative control group gave the net percentage of sensitized animals.
The t-test was used to evaluate differences of the mean body weights between the test substance
group and the control group on Days 0 and 24 ( p= 0.05).

Challenge controls:

The application sites were examined 24 hours after the intradermal induction, 24 hours after the end of the epicutaneous induction exposure and 24 and 48 hours after the end of the epicutaneous challenge exposure.

Positive control substance(s):

yes

Remarks:

Hexyl Cinnamic Aldehide (HCA)

Results and discussion

Positive control results:

Vehicle site: no positive skin reaction in any animal at any reading time.
Substance site: very slight to severe erythema and/or oedema in 7/10 animals 24 and/or 48 hours
after the challenge exposure.
7/10 animals had a "positive skin reaction".

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10% (w/w)

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

no mortality; no significant differences in the mean body weights between the animals of the test substance group and the control group; increased motor activities after beginning of all epicutaneous exposure, which disappear some times later.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% (w/w). No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: no mortality; no significant differences in the mean body weights between the animals of the test substance group and the control group; increased motor activities after beginning of all epicutaneous exposure, which disappear some times later..

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10% (w/w)

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% (w/w). No with. + reactions: 6.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Negative control group:

Vehicle site: No positive skin reaction in any animal at any reading time.

Test substance site: No positive skin reaction in any anima at any reading time.

Test substance group:

Vehicle site: No positive skin reaction in any anima at any reading time

Test substance site: 6/10 animals (60%) has very slight to severe erxthema and or oedema 24 and/or 48 haours after the end of the challenge exposure. 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to the results of this study and to the REGULATION (EC) No 1272/2008, the test substance STI 571 F5 needs to be labelled as a sensitizer,Category 1.