Ichthammol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of study: May 18th, 1984
Final report: June 6th, 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

In this case trademark substance Ichthyol-Sodium (as used as test substance in the toxicity study referred to) is one belonging to the group of dark sulfonated shale oils (SSD) or dark bituminosulfonates. The identifying information "dark" is given as the type of bituminosulfonate in question here is based on a distillate of medium boiling range (dark in color) which is sulfonated under somewhat "severe" temperature conditions that lead to an even darker sulfonated shale oil distillate or bituminosulfonate, respectively. The dark bituminosulfonates comprise the so-called bituminosulfonate "sodium" dark and the bituminosulfonate "ammonium" dark. The production process of dark bituminosulfonates is identical across all refining and intermediate stages. Only the final neutralization with different bases (ammonia or sodium hydroxide) leads to two different salt forms with chemically identical anionic counterpart. In other words, the two salt forms differ with absolutely identical dark bituminosulfonate anions only by the cations "sodium" and "ammonium". The dark bituminosulfonates are therefore grouped together without taking their respective cations into account. Ichthyol-Sodium is manufactured identically to Ichthyol-Ammonium (Ichthammol) in a quality suitable for use in medicinal products. Both substances are obtained from the same manufacturing site.
The common evaluation of dark bituminosulfonates independent on the type of cation has been conducted by different toxicologists and accepted by European authorities (European Medicines Agency) as well as national authorities (German BfArM).

Bituminosulfonate "Sodium" dark is solely used as an active pharmaceutical ingredient in registered medicinal products and as such is exempted from registration under REACH requirements. The "genetic toxicity study in vitro" referred to in this section, for this reason, is not indicated in the substance data set of "Ichthyolic Acid, Sodium Salt" which is based on the available information of bituminosulfonate "sodium" pale (instead of dark). Therefore, no structural reference (read across from supporting substance) and no cross-reference is possible between the substance data sets in this case.

Attached an expert opinion (EXPERT ASSESSMENT OF SODIUM AND AMMONIUM SALTS IN BITUMINOSULFONATES) by Pailer (pharmaceutical chemist) is given who investigated the starting materials for manufacture of Ichthammol for about a decade. He concludes his expert opinion with the statement that it seems readily permissible to combine the (pale and) dark bituminosulfonates into one group without taking into account their respective type of cation.

Furthermore, you can find attached a review article by Cholcha et al. who evaluated the toxicological studies independent on the type of salt (sodium or ammonium) and allocated the conclusions to the toxicological profile of Ichthammol (which is the substance in question here).

As a document published by the European Medicines Agency (EMA) please find attached a Summary Report on "bituminosulfonates, ammonium and sodium salts" (grouped and evaluated together with regard to toxicological effects) that was the outcome of the so-called MRL procedure.

In the European Union, a maximum residue limit (MRL) is the maximum acceptable concentration of a substance that may be found in a food product obtained from an animal that has received a veterinary medicine.

[1] M Pailer, EXPERT ASSESSMENT OF SODIUM AND AMMONIUM SALTS IN BITUMINOSULFONATES, unpublished expert opinion, 1990-05-16
[2] W Cholcha, J Leuschner and F Leuschner, Safety Studies of Dark Sulfonated Shale Oil following Local and Systemic Application, Arzneim.-Forsch./Drug Res. 44(II), 7, 844-849 (1994)
[3] The European Agency for the Evaluation of Medicinal Products (EMA), COMMITTEE FOR MEDICINAL PRODUCTS FOR VETERINARY USE, BITUMINOSULFONATES, AMMONIUM AND SODIUM SALTS, Summary Report, EMEA/MRL/511/98-FINAL, February 1999
[4] The European Agency for the Evaluation of Medicinal Products (EMA), COMMITTEE FOR MEDICINAL PRODUCTS FOR VETERINARY USE, BITUMINOSULFONATES, AMMONIUM AND SODIUM SALTS (Extension to dairy cattle), Summary Report (2), EMEA/MRL/910/04-FINAL, September 2004

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The aim of this study was to evaluate Ichthyol-sodium, batch no. 2/83 for mutagenic activity (gene mutation) in a microbial assay with and without the addition of mammalian metabolic preparations as originally described by AMES et al. (1973, 1975) and revised by MARON and AMES (1983). Performance of study according to 'Nonclinical Laboratory Studies. Good Laboratory Practice Regulations. F.R., Vol. 43, No. 247 dated December 22nd, 1978 and in consideration to the EEC Directive 79/831, Annex V, pages 261 to 265.

Deviations:

not applicable

Principles of method if other than guideline:

Special mutant strains of Salmonella thyphimurium are used, all of them histidine dependent auxotrophs with mutation at a single gene locus (his-). When these organisms are grown on a minimal medium containing only traces of histidine all of them undergo some cell divisions until all histidine is consumed. Within this period of time some organisms spontaneously show reverse mutation back to the histidine-independent wild-type (his+). These organisms grow and form visible colonies on histidine-free medium. After exposure to various classes of mutagens the frequency of reverse mutations to histidine prototrophy (independence) is increased to manifold the value of spontaneous mutations. A possible conversion of the test substance by mammalian enzymes is examined by carrying out the whole test with and without addition of an appropriate enzyme activation mixture .

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Ichthyolic acid, sodium salt

EC Number:

215-671-7

EC Name:

Ichthyolic acid, sodium salt

Cas Number:

1340-06-3

IUPAC Name:

Identification of the UVCB substance by "Chemcial Abstracts Index Name", among others: Ichthyolic Acid, Sodium Salt. No IUPAC name known.

Specific details on test material used for the study:

batch 3/82 from regular production at the registrant's manufacturing site

Method

Test concentrations with justification for top dose:

Preliminary to the main test a dose-range-finding was carried out with one strain: TA 100.
10 dosages were tested with the factor 3.16, between 0.316 µg/plate and 10000 µg/plate as maximum dose level. H2O served as solvent. Plates were prepared as described afore and after 48 h (37°C) observed for the number of revertants (prototrophs) and quality of the background lawn (auxotrophs).

In the pilot study wilh the tester strain TA 100 the concentrations between 0.316 µg to 10000 µg Ichthyol/plate were examined. H2O served as solvent. The dilution factor was 3.16. The dosages were freshly prepared prior to the experiment. No cytotoxicity could be observed up to the highest dosage of 10000 µg/plate. Therefore this dosage was chosen for the highest in the main study.

Vehicle / solvent:

Water (Aqua pro injectione, BRAUN-Melsungen)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation
Sodium azide in H2O (10 µg/pIate): TA 1535, 100
2-nitro-9H-fluorene in DMSO (10 µg/plate): TA 1538, 98
9-amino-acridine in ethanol (50 µg/plate): TA 1537
with metab. act.
2-anthracenamide in DMSO (5 µg/plate): all test strains

Details on test system and experimental conditions:

Autoclaved soft-agar containing 0.61% agar and 0.5% NaCI was melted on the day of the test and 10% of 0.5 mM sterile stock-solution of L-histidine and D-biotin were added before the main test procedure. The agar was kept at 45°C in portions of 2 mI (multi-block heater), then 0.1 ml of salmonella celI-suspension, 0.1 ml of test substance (or solvent/or mutagen) and 0.5 ml of 'S9 mix' (see below) were added. In case of non-activation-assay 'S9 mix' was substituted by 0.5 mI phosphate buffer. This overlay agar mixture was poured on VB-minimal-plates and carefully spread on coded plates. Plates were kept for 48 h at 37°C and the number of revertants on each plate was counted.
This procedure is carried out in 2 independent experiments.

S9 mix (Metabolic Activation System)
Post-mitochondrial fraction (S9 product) from rats induced with Aroclor 7254 was prepared according to MARON & AMES 1983. The S9 was collected from 20 - 30 rats. The pooled lot
was tested for:

- protein content, according to the LOWRY-method
- ability of enzymatic mutagenesis stimulation with the strain TA 1538 and benzo(a)pyrene
- relative P448/P-450 activity

The 59 product is stored in liquid nitrogen and used within three months. 'S9 mix' was freshly prepared on the day of the test containing 10% S9 product according to MARON and
AMES (1983) by adding

- MgCl2 + KCl-salts (sterile stock solution)
- 1 M glucose-5-phosphate (freshly prepared on day of test)
- 0.1 M NADP (freshly prepared on day of test)
- 0.2 M phosphate buffer, pH 7.4 (sterile stock solution)

Afterwards S9 mix was filter-sterilised by using 0.45 pm filters.

Evaluation criteria:

Statistical evaluation of results of the AMES test were still in discussion in 1984. In the testing laboratory a test chemical was considered to have a positive response

- if the number of revertants is significantly increased (rank test) compared to that of spontaneous revertants, reaching at least double the value for TA 98/TA 100, or three times the value for TA 1535, TA 1537 and TA 1538.

- additionally a significant correlation between logarithmic dosages and response (revertants) has to be ascertained. This might implicate the necessity of repeating the test with a narrow dosage.

- positive results have to be reproduced and revertants have to be tested on histidine independence by randomly streaking colonies on histidine free agar.

Statistics:

No details on statistical evaluation given in the original study report apart from reference to a U-test with regard to evaluation of "significant increase of revertants in tester strains with and without activation".

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Complete cytotoxicity could not be observed in TA 98 with or without activation. Some cytotoxicity was observed at the highest dosage of 10000 µg/pIate and the next lower dosage of 3160 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Some cytotoxicity without metabolic activation at the highest dosage of 10000 µg/plate. Otherwise no cytoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Some cytotoxicity without metabolic activation at the highest dosage of 10000 µg/plate. Otherwise no cytoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Some cytotoxicity (not complete) with and without metabolic activation at the highest dosage of 10000 µg/plate. Otherwise no cytoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Some cytotoxicity (not complete) with and without metabolic activation at the two highest dosages of 10000 µg/plate and 3160 µg/plate. Otherwise no cytoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Ichthyol-sodium was tested in 8 dosages between 3.16 and 10000 pg/plate in 5 strains of thyphimurium with and without metabolic activation. Complete cytotoxicitycould not be observed in one of the strains with or without activation, but some cytotoxicity was observed in all strains without metabolic activation and in TA 1537, TA 1538 and TA 98 with metabolic activation at the highest dosage of 10000 pg/pIate. The strains TA 1538 and TA 98 showed slight cytotoxicity at lower dosages of 3160 µg or 1000 µg/plate respectively.
A significant increase of the revertants could not be established in any of the tester strains with and without activation (U-test). A dose-dependent correlation was not found (SPEARMAN's rank coefficient of correlation).
Under the present test conditions there was no indication of mutagenic properties of Ichthyol-sodium.