4'-methoxyacetoacetanilide

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- Rationale for reliability of the study report related to the source substance: Well performed OECD and GLP guideline study (original reliability: 1) - Read across hypothesis: Source and target substance are position isomeric acetoacetanisidides without any other structural differences. Both substances are characterized by similar physical-chemical properties and available data show a consistent toxicity profile. Therfore, it can be reasonably assumed, that acute toxicity, MetHb forming properties and the possible skin sensitizing potential of p-Acetoacetanisidide and o-Acetoacetanisidide are likely to be similar.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum,
Environment: temperature: 22 + 3°C
relative humidity: approx. 30-80%
artificial light: 6.00 a.m. - 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

5.0, 10.0, 25.0 % (w/v)

No. of animals per dose:

4

Details on study design:

Administration and exposure: For determination of the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed on two mice with concentrations of 2.5, 5, 10, and 25 % (w/v). The top dose of the test item is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. 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.

Data and Observations: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). In addition to the sensitising reactions the following observations and data were recorded: mortality/viability once daily, body weights prior to the first application and prior to necropsy, clinical signs (local/systemic) daily from start to the termination of in-life phase.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

Results of the Positive Control Study performed in April 2004

S.I of alpha-Hexylcinnamaldehyde

5 %: 1.9
10 %: 6.1
25 % : 11.5

EC3 = (a-c) [(3-d)/(b-d)] + c = 6.3% (w/v)




Results of the Positive Control GLP Study performed in October 2004

S.I of alpha-Hexylcinnamaldehyde

5 %: 2.0
10 %: 3.0
25 % : 4.9

EC3 = (a-c) [(3-d)/(b-d)] + c = 9.9 % (w/v)

Parameter:

SI

Remarks on result:

other: 5.0 % (w/v): 0.94 10.0 % (w/v): 0.90 25.0 % (w/v): 1.16

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0 % (w/v): 3337 5.0 % (w/v): 3126 10.0 % (w/v): 2993 25.0 % (w/v): 3876

Calculation and Results of individual Data

Vehicle: DMF

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG a)	number of lymph nodes	DPM per lymph node b)	S.I.
-	BG I	0.0	-	-	-	-
-	BG II	0.0	-	-	-	-
-	CG I	3336.7	3336.7	8	417.1
5.0	TG 2	3126.3	3126.3	8	390.8	0.94
10.0	TG 3	2992.5	2992.5	8	374.1	0.90
25.0	TG 4	3876.1	3876.1	8	484.5	1.16

BG = Background (1 ml 5% trichloroaceticacid) 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

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Acetoacet-o-Anisidide TTR was found to be not a skin sensitiser in this assay.

Executive summary:

In the study the test item Acetoacet-o-Anisidide TTR dissolved in DMF was assessed for its possible contact allergenic potential in accordance with OECD test guideline 429, adopted 2002 -04 -24.

For this purpose a local lymph node assay was performed using test item concentrations of 5.0, 10.0, and 25.0 %.

The animals did not show any clinical signs during the course of the study and no cases of mortality observed.

In this study Stimulation Indices (S.I.) of 0.94, 0.90, 1.16 were determined with the test item at concentrations of 5.0, 10.0, and 25.0 % (w/v) in DMF.

The test item Acetoacet-o-Anisidide TTR was found to be not a skin sensitiser in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Discussion of classification or non-classification

Testing of o-acetoacetanisidide for skin sensitising properties in the LLNA revealed negative results. Following read across principles p-acetoacetanisidide is predicted not to be a skin sensitizer.

Read across hypothesis / justification

The purpose of this assessment is to provide justification for read across in order to predict the skin sensitizing potential and MetHb forming properties of the target substance p-acetoacetanisidide (5437-98-9) based on available date coming from the source substances o-acetoacetanisidide (92-15-9).

Source and target substance are position isomeric acetoacetanisidides without any other structural differences. Both substances are characterized by similar physical-chemical properties and available data show a consistent toxicity profile. Therefore, it can reasonably be assumed, that acute toxicity, MetHb forming properties and the possible skin sensitizing potential of p-acetoacetanisidide and o-acetoacetanisidide are likely to be similar.

Data Matrix

	Target Chemical	Source Chemical
CAS #	5437-98-9	92-15-9
CHEMICAL NAME	p-acetoacetanisidide	o-acetoacetanisidide
Other name	4'-methoxyacetoacetanilide N-Acetoacetyl-p-anisidine	2’-methoxyacetoacetanilide N-Acetoacetyl-o-anisidine
Structure	Not displayed	Not displayed
Physical and chemical properties
Purity	99.4 %	99.86 % (w/w)
Physical state	solid	solid
Melting point	116°C	82°C
Decomposition Temp.	>450°C	>450°C
Density	1.242 g/cm^3	1.31 g/cm^3
Vapour pressure (calculate)	< 0.001 Pa at 20°C	0.000036 Pa at 20°C
Log Pow (at 23°C)	0.85	0.91
Water solubility	2.6 g/L	3.24 g/L
Solubility in organic solvents (n-octanol)	> 1 g/L	> 1 g/L
Solubility in organic solvents (DMSO)	> 1 g/L	> 1 g/L
Environmental fate and pathways
Biodegradation	OECD Guideline 302 B: > 97 % after 20 days	OECD Guideline 301 F: Readily biodegradable after 28 days
Ecotoxicological Information
Acute toxicity fish	LC50:     331.7 mg/L LC0:      220.0 mg/L LC100:   500.0 mg/L	LC50:     332 mg/L LC0:      220 mg/L LC100:   500 mg/L
Toxicological Information
Acute Toxicity, oral, rat	LD50: 1755 mg/kg bw	LD50: 1535 mg/kg bw
Acute Toxicity, oral, cat	Read across prediction: MetHb formation	MetHb formation
Skin irritation	Not irritating	Not irritating
Eye irritation	Not irritating	Not irritating
Skin sensitization (LLNA)	Read across prediction: Not sensitising	Not sensitising
Genetic toxicity in vitro (AMES)	negative	negative

Migrated from Short description of key information:
Read across (RA_92-15-9_KEY_429_2004_RCC_850803) LLNA:
Stimulation Indices (S.I.) of 0.94, 0.90, 1.16 were below the threshold of >/= 3 for a positive result and thus lead to the conclusion "not sensitising".

Justification for selection of skin sensitisation endpoint:
most reliable study available from a substance with similar structure

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Stimulation Indices found were below 3 (threshold for a positive result >/= 3) and thus don't meet criteria for classification as skin sensitizing according to REGULATION (EC) No 1272/2008.