4-chlorobenzophenone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Data source

Reference

Reference Type:

publication

Title:

Examination of the Local Lymph Node Assay for Use in Contact Sensitization Risk Assessment

Author:

GERBERICK et.al

Year:

1992

Bibliographic source:

FUNDAMENTAL AND APPLIED TOXICOLOGY, 1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Principles of method if other than guideline:

The objective of the study was to evaluate the utility of the LLNA assay to determine the contact sensitization potential of the test chemical

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid

EC Number:

200-618-2

EC Name:

Benzoic acid

Cas Number:

65-85-0

Molecular formula:

C7H6O2

IUPAC Name:

Benzoic Acid

Test material form:

solid

Details on test material:

Details on test material
- Name of test material : Benzoic acid
- Molecular formula : C7H6O2
- Molecular weight : 122.1224 g/mol
- Smiles notation : c1(ccccc1)C(=O)O
- InChl : 1S/C7H6O2/c8-7(9)6-4-2-1-3-5-6/h1-5H,(H,8,9)
- Substance type: Organic
- Physical state: Solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Labs
- Age at study initiation: 6-9 weeks of age
Animals were housed, fed, and handled in compliance with standards set forth by the U.S. Animal Welfare Act or recommendations in National Institutes of Health “Guide for the Care and Use of Labomtory Animals.” All procedures performed on animals were reviewed and approved by a veterinarian

Study design: in vivo (LLNA)

Vehicle:

other: acetone

Concentration:

5,10,20% test chemical in acetone

No. of animals per dose:

5 animals/ test group

Details on study design:

MAIN STUDY :

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: A chemical was considered positive (a sensitizer) in the local lymph node assay if two criteria were met. First, exposure to at least one concentration of the chemical resulted in a 2-fold or greater increase in [3H]TdR (expressed as dpm) incorporation compared to vehicle-treated control mice.
Second, this mean dpm value was statistically different from vehicle-treated mice (p > 0.0 1). Test materials that failed to cause a greater than a 2-fold elevation of [3H]TdR incorporation were regarded as negative in the local lymph node assay. For ranking purposes, a chemical was considered in the moderate to strong sensitization category if it demonstrated a >30-fold increase in [3H]TdR incorporation over vehicle-treated mice. A chemical in the 2- to 30-fold range of increased [3H]TdR incorporation was classified as a weak to- moderate sensitizer.


TREATMENT PREPARATION AND ADMINISTRATION:12.5 microliters of test material or vehicle applied to each side of both ears (25 microliter total/ear). Five animals per test group were used. Eighteen to 24 hr after the fourth induction treatment, 20 microCi of [methyl-[3H]thymidine ([3H]TdR; 5.0 curies/mmol sp act, Amersham Corp., IL), in 0.25 ml of phosphate-buffered saline (PBS; GIBCO, NY), was injected into the tail vein of each mouse. The mice were euthanized 5 hr after [‘H]TdR injection. The bilateral auricular lymph nodes were excised and pooled for each mouse.
A single cell suspension was prepared from the lymph nodes of each mouse by gently rubbing the nodes through a nylon mesh filter (100 micrometer pore size, The Spectra Co., CA). The cell suspensions were washed with 10 ml PBS, re-suspended in 1 ml PBS and a 20-/11 sample of the cell suspension taken for cell number determination using an automated cell counter (Coulter Model ZM; Coulter Electronics, Inc., FL).
Following a second wash in PBS, the cell pellet was re- suspended in 3 ml of 5% trichloroacetic acid (TCA; Sigma) and left overnight (- 18 hr) at 0-4°C.
The samples were centrifuged, the supernatant was decanted, and the pellet was re-suspended in 2 ml of 5% TCA. The cells were transferred to vials containing 10 ml liquid scintillation cocktail (Ready Safe; Beckman Instruments, CA). The ‘H disintegrations per minute (dpm) were determined by counting for 5 to 10 min on a liquid scintillation counter (Beckman Model LS5000TD, Beckman Instruments, Inc., CA).

Positive control substance(s):

not specified

Statistics:

A chemical was considered positive (a sensitizer) in the local lymph node assay if two criteria were met. First, exposure to at least one concentration of the chemical resulted in a 2-fold or greater increase in [3H]TdR (expressed as dpm) incorporation compared to vehicle-treated control mice.Second, this mean dpm value was statistically different from vehicle-treated mice (p > 0.0 1). Test materials that failed to cause a greater than a 2-fold elevation of [3H]TdR incorporation were regarded as negative in the local lymph node assay. For ranking purposes, a chemical was considered in the moderate to strong sensitization category if it demonstrated a >30-fold increase in [3H]TdR incorporation over vehicle-treated mice. A chemical in the 2- to 30-fold range of increased [3H]TdR incorporation was classified as a weak to- moderate sensitizer.
A chemical was considered positive (a sensitizer) in the local lymph node assay if two criteria were met. First, exposure to at least one concentration of the chemical resulted in a 2-fold or greater increase in [3H]TdR (expressed as dpm) incorporation compared to vehicle-treated control mice. Second, this mean dpm value was statistically different from
vehicle-treated mice (p > 0.0 1). Test materials that failed to cause a greater than a 2-fold elevation of [3H]TdR incorporation were regarded as negative in the local lymph node assay. For ranking purposes, a chemical was considered in the moderate to strong sensitization category if it demonstrated a >30-fold increase in [3H]TdR incorporation over vehicle-treated mice. A chemical in the 2- to 30-fold range of increased [3H]TdR incorporation was classified as weak to- moderate sensitizer.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

other: dpm

Value:

4.2

Test group / Remarks:

5% test chemical in acetone

Remarks on result:

other: not sensitizing

Results 2

Parameter:

other: dpm

Value:

5

Test group / Remarks:

10% test chemical in acetone

Remarks on result:

other: not sensitizing

Results 3

Parameter:

other: dpm

Value:

4.27

Test group / Remarks:

20% test chemical in acetone

Remarks on result:

other: not sensitizing

Cellular proliferation data / Observations:

The test chemical failed to stimulate a greater than 2-fold response

Any other information on results incl. tables

Measurement of Lymphocyte proliferation in Murine Local Lymph Node assay

	Test concentration	Mean cell number (*10-6)	Mean dpm (*10-2)	Dpm fold increase
	Acetone	4.91± 0.9	5.42± 0.8	-
	5%	3.84± 0.3	4.20± 0.6	0.8
	10%	3.93± 0.4	5.00± 0.7	0.9
	20%	3.66± 0.6	4.27± 0.6	0.8

 

Applicant's summary and conclusion

Interpretation of results:

other: not sensitizing

Conclusions:

The mean dpm values for 5%,10% and 20% test chemical in acetone were 0.042, 0.05 and 0.0427 respectively. The test chemical failed to stimulate a greater than 2-fold response.Hence, the test chemical was considered to be not sensitizing to mice skin.

Executive summary:

The objective of the study was to evaluate the utility of the LLNA assay to determine the contact sensitization potential of the read across substance Benzoic acid (CAS no.: 65 -85 -0, E.C. no.: 200 -618 -2). 5, 10, 20% test chemical in acetone was used as test concentrations.Female CBA/J mice 8-9 weeks old were used for the study. 12.5 microliters of test material or vehicle applied to each side of both ears (25 microliter total/ear). Five animals per test group were used. Eighteen to 24 hr after the fourth induction treatment, 20 microCi of [methyl-[3H]thymidine ([3H]TdR; 5.0 curies/mmol sp act, Amersham Corp., IL), in 0.25 ml of phosphate-buffered saline (PBS; GIBCO, NY), was injected into the tail vein of each mouse. The mice were euthanized 5 hr after [‘H]TdR injection. The bilateral auricular lymph nodes were excised and pooled for each mouse. A single cell suspension was prepared from the lymph nodes of each mouse by gently rubbing the nodes through a nylon mesh filter (100 micrometer pore size, The Spectra Co., CA). The cell suspensions were washed with 10 ml PBS, re-suspended in 1 ml PBS and a 20-/11 sample of the cell suspension taken for cell number determination using an automated cell counter (Coulter Model ZM; Coulter Electronics, Inc., FL).Following a second wash in PBS, the cell pellet was re- suspended in 3 ml of 5% trichloroacetic acid (TCA; Sigma) and left overnight (- 18 hr) at 0-4°C. The samples were centrifuged, the supernatant was decanted, and the pellet was re-suspended in 2 ml of 5% TCA. The cells were transferred to vials containing 10 ml liquid scintillation cocktail (Ready Safe; Beckman Instruments, CA). The ‘H disintegrations per minute (dpm) were determined by counting for 5 to 10 min on a liquid scintillation counter (Beckman Model LS5000TD, Beckman Instruments, Inc., CA). A chemical was considered positive (a sensitizer) in the local lymph node assay if two criteria were met. First, exposure to at least one concentration of the chemical resultedin a 2-fold or greater increase in [3H]TdR (expressed as dpm)incorporation compared to vehicle-treated control mice.Second, this mean dpm value was statistically different from vehicle-treated mice (p > 0.0 1). Test materials that failed to cause a greater than a 2-fold elevation of [3H]TdR incorporation were regarded as negative in the local lymph node assay. For ranking purposes, a chemical was considered in the moderate to strong sensitization category if it demonstrated a >30-fold increase in [3H]TdR incorporation over vehicle-treated mice. A chemical in the 2- to 30-fold range of increased [3H]TdR incorporation was classified as a weak to- moderate sensitizer. The mean dpm values for 5%,10% and 20% test chemical in acetone were 0.042, 0.05 and 0.0427 respectively. The test chemical failed to stimulate a greater than 2-fold response. Hence, the test chemical was considered to be not sensitizing to mice skin.