Butanamide, 2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-, N,N'-bis(4-chloro-2,5-dimethoxyphenyl and 2,4-xylyl) derivs.

Endpoint summary

Description of key information

Testing for sensitizing properties of PY176 was performed in female guinea pigs according to the method of Magnusson & Kliggman. Intradermal induction was perfored using 1% test item in hydroxyethyl-cellulose (1% in water). Dermal induction and challenge treatment were carried out with 25% test item in hydroxyethyl-cellulose (1% in water) (corresponding to 14.7% P.Y. 176 in hydroxyethyl cellulose - 1% in water ). Based on the results of this study the test item showed no evidence of sensitizing properties. Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.

In the study the analogue substance PY13 suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5 and 10% (w/v).

The animals (4 female mice/dose grouop) did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.71, 1.32 and 2.26 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in propylene glycol, respectively. The results obtained with the positive control confirmed the validity of the test.

The test item was not a skin sensitiser in this assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 AUG 2006 to 15 AUG 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study (OECD TG 429)

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to Chemikaliengesetz and Directive 88/320/EEC

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 7-8 weeks (beginning of acclimatization)
- Weight at study initiation: mean: 19.2 g (17.1-20.8)
- Housing: individually, Makrolon Type I cages
- Diet: pelleted standard diet (Harlan Winkelmann, Borchen), ad libitum
- Water: tap water, ad libitum
- Acclimatization: yes (acclimatization period not given)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3
- Humidity (%): 30-85%
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Vehicle:

propylene glycol

Concentration:

0%, 2.5%, 5%, 10% (w/v)

No. of animals per dose:

4 females per dose group
2 females in the pre-test

Details on study design:

RANGE FINDING TESTS:
- non GLP
- Compound solubility: 10% (w/v) suspension in propylene glycol was the highest technically applicable concentration; higher concentrations could also not be achieved with other vehicles
- Irritation: no irritation effects were observed at these concentrations after a single application
- Lymph node proliferation response: no data


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1: exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index
2: data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression


TREATMENT PREPARATION AND ADMINISTRATION:
- individual preparation of weight volume dilutions using a magnetic stirrer as homogenizer
- test item preparations were made freshly before each dosing occasion
- 25 µl were spread over the entire dorsal surface of each ear lobe once daily for three consecutive days

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

- calculation of mean values and standard deviations for body weight

Positive control results:

Stimulation indices of 2.30, 4.31 and 11.21 were determined with the positive control substance at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil (4+1). An EC3 value of 6.7% (w/v) was calculated.

Key result

Parameter:

SI

Value:

2.26

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

17.1

Test group / Remarks:

2,5%

Remarks on result:

other: Stimulation indices were all below 3. The following SI were calculated: 2.5% test item: 1.71 5% test item: 1.32 10% test item: 2.26

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

There was a dose dependent increase in the dpm, which were measured for the pooled lymph nodes of each treatment group (8 lymph nodes per dose group): Background: 17.56 or 20.36 dpm Control group: 3102.24 dpm 2.5% test item: 5291.91 dpm 5% test item: 4100.26 dpm 10% test item: 6990.34 dpm

Key result

Parameter:

SI

Value:

1.32

Test group / Remarks:

5%

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. The body weight of the animals was within the normal range. Calculation of the EC3 value was not performed, because no test concentration produced a SI of 3 or higher.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test substance was not sensitising in this LLNA in concentrations up to 10% (w/v) in propylene glycol, the highest technically achievable concentration.

Executive summary:

In the study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5 and 10% (w/v).

The animals (4 female mice/dose grouop) did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.71, 1.32 and 2.26 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in propylene glycol, respectively. The results obtained with the positive control confirmed the validity of the test.

The test item was not a skin sensitiser in this assay. Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 APR 1998 to 29 MAY 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study (OECD TG 406)

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

At the time of study performance the applied test method was recommended by OECD as a standard method. OECD TG 429 was not yet available.

Species:

guinea pig

Strain:

other: Pirbright HsdPoc:DH

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Weight at study initiation: mean: 369 g
- Housing: in groups of 5 animals, Macrolon cages Type 4
- Diet: ssniff Ms-H (V2233), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12 hrs/12hrs

Route:

intradermal and epicutaneous

Vehicle:

other: control group: sesame oil DAB 10; treatment group: hydroxyethyl-cellulose (1 % in water)

Concentration / amount:

1%: intradermal induction
25%: dermal induction and challenge

Route:

epicutaneous, occlusive

Vehicle:

other: control group: sesame oil DAB 10; treatment group: hydroxyethyl-cellulose (1 % in water)

Concentration / amount:

1%: intradermal induction
25%: dermal induction and challenge

No. of animals per dose:

10 animals per treatment group
5 animals in control group
2 animals for determination of the tolerance of the intradermal injections
3 animals for determination of the primary non-irritant concentration

Details on study design:

RANGE FINDING TESTS:
- Determination of primary non-irritation concentration in a dermal occlusive test: application of 0.5 ml of a 1%, 5% or 25% test item preparation in hydroxyethyl-cellulose (1% in water) to the flanks, occlusive exposure for 24 hours; reading 24 hours after patch removal.

-Determination of the tolerance of the intradermal injections: application of 0.1 ml of 0.2%, 1.0% or 5.0% preparation in hydroxyethyl-cellulose (1% in water) at the dorsal area; examination for local tolerance: 24, 48, 72 and 96 hours after administration.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 pairs of indtradermal injections and one dermal exposure
- Site: dorsal area
- Frequency of applications: day 1: intradermal induction; day 8: start of dermal induction (lasting for 48 hours, occlusive)
- Concentrations:

Treatment group:
intradermal: 0.1 ml of 50% FCA or 1% preparation in hydroxyethyl-cellulose (1% in water) or 1% preparation in 50% FCA; dermal: 0.5 ml of 25% preparation in hydroxyethyl-cellulose (1% in water)

Control group:
intradermal: 0.1 ml of 50% FCA or sesam oil or sesam oil:50% FCA (1+1); dermal: 0.5 ml of hydroxyethyl-cellulose (1% in water)


B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day of challenge: day 22 of the study
- Exposure period: 24 hours, occlusive
- Site: left flank
- Concentrations: 25% preparation of test item in hydroxyethyl-cellulose (1% in water)
- Evaluation (hr after challenge): 24 and 48 hours after removal of the patch (day 24 and 25 of the study)

Challenge controls:

- none

Positive control substance(s):

yes

Remarks:

Benzocain

Positive control results:

Intradermal induction: 1 % Benzocain in sesame oil
first challenge (day 22) with 25% Benzocain in sesame oil: 20% (2 of10) of the animals showed a positive reaction at the first reading after 24 hrs
second challenge (day 29) with 25% Benzocain in sesame oil: 50% (5 of 10) of the animals showed a positive reaction at the first reading after 24 hrs

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% in hydroxyethyl-cellulose (1% in water)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No clinical signs of toxicity, no impairment of body weight gains.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% in hydroxyethyl-cellulose (1% in water). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical signs of toxicity, no impairment of body weight gains..

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% in hydroxyethyl-cellulose (1% in water)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No clinical signs of toxicity, no impairment of body weight gains.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% in hydroxyethyl-cellulose (1% in water). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical signs of toxicity, no impairment of body weight gains..

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% in hydroxethyl-cellulose (1% in water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No clinical signs of toxicity, no impairment of body weight gains.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% in hydroxethyl-cellulose (1% in water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No clinical signs of toxicity, no impairment of body weight gains..

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% in hydroxyethyl-cellulose (1% in water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No clinical signs of toxicity, no impairment of body weight gains.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% in hydroxyethyl-cellulose (1% in water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No clinical signs of toxicity, no impairment of body weight gains..

- Intradermal Induction: Intradermal injections with FCA (with and without test substance) caused severe erythema and oedemas as well as indurations and encrustations. Administration sites treated with test substance showed slight erythema and oedema. Sesame oil did not induce irritations.

- Dermal Induction: After removal of the patches severe erythema and oedema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with FCA. Administration sites treated with test substance or sesame oil showed no signs of irritation.

- Challenge: In all cases the treated skin areas were discoloured slight yellow. No signs of irritation were observed in the control and treatment group 24 and 48 hours after removal of the occlusive bandage.

Interpretation of results:

not sensitising

Conclusions:

The test substance was not sensitising in concentrations up to 25% in hydroxyethyl cellulose (1% in water) (corresponding to 14.7% P.Y. 176 in hydroxyethyl cellulose - 1% in water ) in this guinea pig maximization test. This test was considered sufficient to fulfil the information requirements.

Executive summary:

Testing for sensitizing properties of the test item was performed in female guinea pigs according to the method of Magnusson & Kliggman. Intradermal induction was perfored using 1% test item in hydroxyethyl-cellulose (1% in water). Dermal induction and challenge treatment were carried out with 25% test item in hydroxyethyl-cellulose (1% in water) (corresponding to 14.7% P.Y. 176 in hydroxyethyl cellulose - 1% in water ). Based on the results of this study the test item showed no evidence of sensitizing properties. Therefore, the test item has not to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.

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

No classification

The test item did no cause sensitising effects in a maximisation test in Guinea pigs. The close analogue PY13 did not reveal sensitising properties in an LLNA in mice.