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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-08-26 to 2004-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study documented and performed according to GLP standards and in compliance with OECD Guideline 429: Skin sensitization: Local Lymph Node Assay
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2'-methylacetoacetanilide
EC Number:
202-267-0
EC Name:
2'-methylacetoacetanilide
Cas Number:
93-68-5
Molecular formula:
C11H13NO2
IUPAC Name:
N-(2-methylphenyl)-3-oxobutanamide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identity: Acetoacet-o-Toluidide
Aggregate state at RT: Solid
Color: White
Storage: At RT, dry. dark
Expiry date: July 25, 2006

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Strain: CBA/CaOlaHsd
Source: Harlan Netherlands
Number of animals for pre-test: 2 females
Number of animals for main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Age: 8-12 weeks (beginning of acclimatization)
Identification: Single caging.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 10, 25 % (in DMF)
No. of animals per dose:
4 females
Details on study design:
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test andcontrol raw data.
The mean values and standard deviations were calculated in the body weight tables.

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.

Results and discussion

Positive control results:
Positive control substance: α-Hexylcinnamaldehyde
Vehicle: Acetone/olive oil

Test item concentration %
(w/v) S.I.
Group 2 5 (a) 1.9 (b)
Group 3 10 (c) 6.1 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 6.3 % (w/v)

EC3 = Estimated concentration for a S.I. of 3.
a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below the S.I.
value of 3 on the LLNA dose response plot.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table below

Any other information on results incl. tables

Results:

Vehicle: DMF

Test item concentration % (w/v)

Group

Measurement DPM

DPM-BG a)

Number of Lymph Nodes

DPM per Lymph Node b)

S.I.

---

BG I

0.0

---

---

---

---

---

BG II

0.0

---

---

---

---

---

CG 1

3336.7

3336.7

8

417.1

 

5.0

TG 2

3776.3

3776.3

8

472.0

1.13

10.0

TG 3

3877.1

3877.1

8

484.6

1.16

25.0

TG 4

3320.4

3320.4

8

415.1

1.00

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 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item Acetoacet-o-Toluidide was found to be not a skin sensitiser.
Executive summary:

The LLNA study was documented and performed in year 2004 according to GLP standards and in compliance with OECD Guideline 429: Skin sensitization: Local Lymph Node Assay. As test system Mice, strain CBA/CaOlaHsd was used. Upon sponsor's request the data of and additional validation- / positive control study performed under GLP are included in the study report.

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. The body weights of the animals, recorded prior to the 1st application and prior to necropsy was within the range commonly recorded for animals of this strain and age.

In this study stimulation indices of 1.13, 1.16, and 1.00 were determined with the test item at concentrations of 5 %, 10 %, and 25 % (w/v) in vehicle, respectively. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared to the concurrent control, as indicated by the stimulation index.

The estimated concentration of test item required to produce an S.I. of 3 is referred to as the EC3 value. In this study the EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Therefore, the test item Acetoacet-o-Toluidide was not found to be a skin sensitiser.

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