3-amino-4-chlorobenzoic acid

Skin sensitisation

Administrative data

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

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read across substance, only limited data on test substance and animals or environmental conditions available, substance preparation and analysis not described

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

standard protocol described previously by Kimber and Basketter, 1992

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

para-aminobenzoic acid

IUPAC Name:

para-aminobenzoic acid

Constituent 2

Reference substance name:

4-aminobenzoic acid

EC Number:

205-753-0

EC Name:

4-aminobenzoic acid

Cas Number:

150-13-0

IUPAC Name:

4-aminobenzoic acid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (Laboratories A and B; Harlan Olac, Bicester, Oxfordshire, UK) or female CBA/JHsd mice (Laboratories C, D and E; Harlan Sprague Dawley, Inc., Frederick, MD, USA)
6-12 weeks old
housed under standard conditions
Food and tap water were available ad libitum.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 1, 2.5, 5, 10%

No. of animals per dose:

4-5

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of mice (n = 4 or 5) were treated by topical application with 25 microliter of various concentrations of the test material or with vehicle alone on the dorsum of both ears. Treatments were performed daily for three consecutive days and then the mice were rested for 2 days prior to analysis.
On the sixth day, the mice were injected intravenously via the tail vein with 250 microliter of phosphate buffered saline (PBS) containing 20 microCi of tritiated methyl thymidine ( 3H-TdR; specific activity 2 Ci/mmol; Amersham International, Amersham, Bucks, UK).
Five h later mice were sacrificed and the draining auricular lymph nodes excised and pooled for each experimental group. Single cell suspensions of LNC were prepared by gentle mechanical disaggregation through 200-mesh stainless steel gauze. Pooled LNC were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. Approximately 12 h later the samples were pelleted by centrifugation, resuspended in 1 ml 5% TCA and transferred to 10 ml of scintillation fluid (Optiphase MP, LKB Scintillation Products, FSA Laboratory Supplies, UK).
Incorporation of 3HTdR was measured by gamma-scintillation counting and expressed as disintegrations per minute (dpm) per node by dividing the total dpm per group by the number of nodes.
Stimulation indices (SI) for each experimental group were determined as the increase in 3H-TdR incorporation relative to vehicle-treated controls.
In three of five laboratories modiefied tests were performed:
4 days treatment, no pooling or
[125-iododeoxyuridine instead of thymidine and no pooling

Positive control substance(s):

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

Statistics:

In Laboratories C, D and E, in which isotope incorporation was determined for individual mice, a mean dpm value f S.E. was calculated for each experimental group. A stimulation index was derived for each experimental group by dividing the mean dpm of the experimental group by the mean dpm of the vehicle control group. For statistical analyses, the mean dpm values for each test chemical concentration treatment group were initially normalized by obtaining their log value. Bartlett’s test (Bartlett, 1937) was then used to examine the data for homogeneity of the within-chemical treatment variance. When analysis of variance revealed significant differences in parametric data, experimental groups were compared with vehicle-treated controls using Dunnett’s t test (Dunnett, 1955).
For non-parametric data, a Kruskal-Wallis test (Kruskal and Wallis, 1952) followed by Dunn’s multiple comparison procedure (Dunn, 1964) was used. Groups differing from vehicle-treated controls at the level of P I 0.01 were considered significant. Jonckheere’s trend analysis (Gross and Clark, 1975) was used to test for dose-dependent effects at a significance level of P I 0.01.
Stimulation indices from each of the live participating Laboratories were compared by analysis of covariance separately for each chemical. Differences in overall stimulation indices obtained and in the relationship between stimulation indices and dose amongst the participating Laboratories were evaluated. Estimates of the test material concentration required to produce a stimulation index of 3 were calculated for DNCB, HCA, oxazolone, isoeugenol, eugenol and SLS using fitted quadratic regression analyses.

Results and discussion

Positive control results:

3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Exposure Zeneca (A) Unilever (B) Procter & Gamble (C) IITRI (D) DuPont (E) concentration % vehicle 0.5 0.9 1.2 1.1 1.6 1.1 1 1.0 1.2 1.1 0.8 0.6 2.5 1.4 1.1 1.2 0.9 0.7 5 1.1 1.6 1.1 0.7 0.8 10 1.0 1.4 1.0 0.7 0.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Exposure Zeneca (A) Unilever (B) Procter & Gamble (C) IITRI (D) DuPont (E) concentration % vehicle 453 438 90 139 59 0.5 399 546 101 223 67 1 457 536 98 116 37 2.5 626 486 104 121 39 5 519 682 100 91 46 10 452 593 86 97 35

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information