Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Data source

Reference

Reference Type:

publication

Title:

The comet assay with 8 mouse organs: results with 39 currently used food additives

Author:

Yu F. Sasaki, Satomi Kawaguchi, Asako Kamaya, Miyuki Ohshita, Kazumi Kabasawa, Kayoko Iwama, Kazuyuki Taniguchi, Shuji Tsuda

Year:

2002

Bibliographic source:

Mutation Research 519 (2002) 103-119

Materials and methods

Principles of method if other than guideline:

Comet assay with 8 organs in mice

GLP compliance:

no

Type of assay:

other: Comet-assay

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

EC Number:

223-339-8

EC Name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Cas Number:

3844-45-9

Molecular formula:

C37 H34 N2 Na2 O9 S3

IUPAC Name:

disodium 2-({4-[ethyl(3-sulfonatobenzyl)amino]phenyl}{4-[ethyl(3-sulfonatobenzyl)iminio]cyclohexa-2,5-dien-1-ylidene}methyl)benzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

Test substance: Brilliant Blue FCF
Test substance: Neptune Blue BRA concentrate

Specific details on test material used for the study:

Identity: Brilliant Blue FCF, CAS 3844-45-9, Colour index CI-42090
-supplier: Tokyo Kasei Kogyo Industry Ltd, Tokyo, Japan;

Test animals

Species:

mouse

Strain:

other: ddY mice

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan SLC Co., Shizuoka, Japan
- Age at study initiation: 7 weeks
- Weight at study initiation: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: No data

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

physiological saline

Duration of treatment / exposure:

Single treatment, sacrifice after 3h and 24h

Frequency of treatment:

single treatment

Post exposure period:

3 and 24h

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent no treatment

Positive control(s):

The study did not include an established positive control. Positive findings were reported for a number of the 39 tested food additives.

Examinations

Tissues and cell types examined:

Stomach, Colon, Liver, Kidney, Bladder, Lung, Brain, Bone marrow

Details of tissue and slide preparation:

The liver, kidney, Jung, and brain were minced, suspended in 4 ml chilled homogenizing solution (pH 7 .5) containing 0.075 M NaCI and 0.024 M Na2EDTA, and then homogenized gently using a Potter-Elvehjem type
homogenizer at 500-800 rpm, in ice.
The glandular stomach, colon, and urinary bladder were opened and rinsed with physiological saline; the mucosa was scraped into 4 ml chilled homogenizing buffer and homogenized gently using a Potter-Elvehjem type
homogenizer at 500-800 rpm, in ice. To obtain nuclei, the homogenate was centrifuged at 700 x g for 10 min at 0 °C, and the precipitate was re-suspended in chilled homogenizing buffer at 1 g organ weight/ml.

Seventy-five microliters agarose GP-42 was quickly layered on a slide (Matsunami Glass Ind. Ltd., Osaka, Japan) coated with agarose GP-42 and covered with another slide. The slide sandwiches were placed horizontally to allow the agarose to solidify. The nucleus suspension was mixed 1: 1 (v/v) with 2%, 45 °C, agarose LGT, and 75 μ! of the nucleus mixture was quickly layered in the same manner after removal

The length of the whole comet ("length") and the diameter of the head ("diameter") were measured for 50 nuclei per organ
per animal. We calculated migration as the difference between length and diameter for each of 50 nuclei.
Mean migration of 50 nuclei from each organ was calculated for each individual animal.

Evaluation criteria:

An increase in DNA damage was indicated by a statistically significant increase in DNA migration

Statistics:

The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P value less
than 0.05 was considered statistically significant.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

other: Positive findings were observed for other substances in this study.

Any other information on results incl. tables

Migration (μm, mcan ± S.E.M. of four animals)

	Time after dosing	stomach	colon	liver	kidney	bladder	lung	Brain	Bone marrow
control	-	5.71 ± 0.52	5.42 ± 0.87	2.35 ± 0.49	2.27 ± 0.62	5.45 ± 0.74	2.84 ± 0.19	0.95 ± 0.36	1.23 ± 0.45
2000 mg/kg bw	3h	4.03 ± 0.60	3.49 ± 1.56	5.39 ± 1.36	4.63 ± 0.88	4.57 ± 0.97	4.62 ± 0.54	2.14 ± 0.98	0.65 ± 0.44
2000 mg/kg bw	24h	5.75 ± 0.96	4.77 ± 1.85	1.09 ± 0.97	5.27 ± 1.35	5.21 ± 0.81	4.25 ± 1.90	2.50 ± 1.18	4.57 ± 1.35

Applicant's summary and conclusion

Conclusions:

A single treatment of mice with 2000 mg/kg bw did not result in DNA damage as indicated by comet formation.