2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Endpoint summary

Description of key information

Skin sensitising potential of Pigment Yellow 74 was tested in two Local Lymph Node Assays (LLNA) according to OECD test guideline 429. In the assay selected as key study S.I. of 0.7, 0.6 and 1.4 were determined with the test item at concentrations of 1%, 2.5% and 5% in acetone:olive oil (4:1 v/v). This result is supported by observations in the second study, where the test item was suspended in propylene glycol at concentrations of 2.5%, 5%, and 10% (w/v). Stimulation Indices of 0.45, 1.02 and 2.18 were determined, respectively.

In both assays no EC3 value could be calculated as all S.I. values were below 3.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2006 to 12 December 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and OECD Principles of Good Laboratory Practice

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: 18.3 g to 22.5 g
- Housing: single caging, Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

propylene glycol

Concentration:

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

No. of animals per dose:

4 animals per treatment group, 5 animals in the control group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
To determine the highest non-irritant test concentration or the highest technically
applicable concentration, a pretest was performed under RCC-CCR non-GLP study
number 1044303 in two mice. The data showed that the highest test item concentration,
which could be technically used was a 10 % suspension. Even warming and sonicating
could not achieve a higher concentration. With other vehicles used, e.g actone:olive oil
(4+1), DMF, DMSO, or ethanol:deionised water (7+3), higher concentrations could also not
be achieved. Upon sponsors consent 10 % was used as the highest concentration.
- Irritation:
At this concentration the treated mouse did not show any toxic symptoms. Due to the colour of
the test item irritation of the ear skin could not be observed.
- Lymph node proliferation response: not evaluated


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled (4 animals/group) LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
# First, that exposure to at least one concentration of the test item resulted in an
incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as
indicated by the stimulation index.
# Second, that the data are compatible with a conventional dose response, although
allowance must be made (especially at high topical concentrations) for either local
toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
Four female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

Positive control substance(s):

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

Statistics:

-

Positive control results:

Test item concentration % (w/v) Group Measurement DPM DPM-BG a) Number of lymph nodes DPM per lymph node b) S.I.
- BG I 197.50 - - - -
- BG II 22.03 - - - -
- CG 1 4049.44 3939.7 8 492.5
5 TG 1 8152.94 8043.2 8 1005.4 2.04
10 TG 2 24974.40 24864.6 8 3108.1 6.31
25 TG 3 49155.30 49045.5 8 6130.7 12.45
BG = Background (1 ml 5% trichloroacetic acid) in duplicate
CG = Control Group (Vehicle: acetone:olive oil (4+1))
TG = Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

EC3 = (a-c) [(3-d)/(b-d)] + c = 6.1% (w/v)

Key result

Parameter:

SI

Value:

0.45

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

1.02

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

2.18

Test group / Remarks:

10%

Parameter:

SI

Remarks on result:

other: The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Table below

Test item concentration% (w/v)   Measurement dpm     Calculation      Result     dpm - BG a) number of lymph nodes dpm per lymph node b)  S.I.   -- BG I  21.70  -- -- -- --  -- BG II   13.65  -- -- --  --   --  CG 1  5794.44  5776.8  10 577.7  -- 2.5  TG 1  2089.28  2071.6  8 259.0 0.45 5  TG 2  4715.50 4697.8  8 587.2 1.02  10  TG 3  10070.60  10052.9  8 1256.6  2.18 BG = Background (1 ml 5% trichloroacetic acid) in duplicate CG = Control Group TG = Test Group S.I. = Stimulation Index a) = The mean value was taken from the figures BG I and BG II b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was determined by dividing the measured value by the number of lymph nodes pooled The EC3 value could not be calculated, since all SI's are below. No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Body weights were within the normal range.

Interpretation of results:

not sensitising

Conclusions:

The test item was found to be not a skin sensitiser in the LLNA when tested at up to 10% in propylene glycol.

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 %. The animals 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 0.45, 1.02, and 2.18 were determined with the test item at concentrations of 2.5, 5, and 10 % in propylene glycol, respectively. Since all S.I. were below 3 no EC3 value could be derived. The test item was not a skin sensitiser in this assay.

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

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

Pigment Yellow 74 did not show skin sensitising properties in two LLNA studies according to the current OECD guidelines. Allergic skin reactions or case reports of acute contact dermatitis to Pigment Yellow 74 have not been described in the literature. Therefore, Pigment Yellow 74 does not have to be classified as a skin sensitizer according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).