Registration Dossier

Administrative data

Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of study: March 22nd, 1995
Termination of study: March 23rd, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Ichthammol
EC Number:
232-439-0
EC Name:
Ichthammol
Cas Number:
8029-68-3
Molecular formula:
unspecified
IUPAC Name:
Ichthammol (UVCB substance)
Test material form:
liquid: viscous
Details on test material:
sample from a batch from regular production released for use as an active pharmaceutical ingredient in medicinal products.
Specific details on test material used for the study:
The test substance was taken from a regular production batch of the registrant's manufacturing site (Ichthyol batch no. R 954623). Ichthyol is a trademark name for Ichthammol. A certificate of analysis is part of the study report. Release of raw material was done according to provisions of different pharmacopoeias (ÖAB, PH.EUR and DAB 10, respectively).

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
Test solutions consisted of inoculation medium, stock solutions and test substance solution. For preparation of the different solutions aqua dest. was used.

Test organisms

Test organisms (species):
Pseudomonas putida
Details on inoculum:
One day old stock cultures of Pseudomonas putida served as inocula for preliminary cultures. Using aseptic techniques, suspensions of bacteria in preliminary culture medium (stock solution I, III, IV; 2.5 ml stock solution I, 2.5 ml stock solution III, 5.0 ml stock solution IV; see below) were diluted to a concentration corresponding a turbidity value of TU/F = 10 (turbidity units formazin).

Stock solution I
10.0 g sodium nitrate, NaNO3;
2.4 g dipotassium hydrogen phosphate, K2HPO4
1.2 g potassium dihydrogen phosphate, KH2PO4
1.0 g yeast extract
in 500 ml aqua dest.

Stock solution II
10.0 g sodium nitrate
2.4 g dipotassium hydrogen phosphate
1.2 g potassium dihydrogen phosphate
in 500 ml aqua dest.

Stock solution III
44.0 g D(+)-glucose-monohydrate
in 500 ml aqua dest.

Stock solution IV
0.01 g ferrous citrate, w(Fe) = 19%
4.0 g magnesium sulphate, heptahydrate
in 1000 ml aqua dest.

250-ml Erlenmeyer flasks were each filled with 100 ml of suspension. The bacteria were incubated at 20°C - 21°C for about 7 hours. Thereafter the cultures were mixed and adjusted with further preliminary culture medium to an extinction value of 0.168 (TU/F = 50 ± 1). This suspension served as inoculation medium for the test.

Four parallel dilution series in 250-ml Erlenmeyer flasks with sterile stoppers were prepared with a final volume of 100 ml test medium each. Three series were inoculated with bacteria. The fourth series was not inoculated and served as a blank.
The final volume consisted of 10 ml of the inoculation medium (or 10 ml of preliminary culture medium for the fourth series which was not inoculated), 10 ml of stock solutions (2.5 ml stock solution II, 2.5 ml stock solution III, 5.0 ml stock solution IV; cf Appendix 1) and 80 ml of the test substance solution. The dilution factor was 2.0. The bacterial concentration at start corresponded to a turbidity value of TU/F = 5. The pH was 6.88 to 6.95.
Four control culture flasks with 80 ml sterile water, 2.5 ml stock solution II, 2.5 ml of stock solution III, 5 ml of stock solution IV and 10 ml of the prepared bacterial solution (3 flasks) or 10 ml of preliminary culture medium (1 flask) were also included.
Both inoculated and non-inoculated (see above) dilution series as well as the control culture flasks were left at 20°C - 21°C in the dark for 16 ± 1 hours (shaker; 75 - 85 rpm).

After termination of the test period the extinction of the monochromatic light at 436 nm was measured in a 10-mm cell with samples from the four test substance flasks per concentration and the control flasks. Since turbidity occurred in the dilution series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series were used as photometric blank values for the turbidity of the inoculated dilution series.

In a preliminary test the bacteria culture was exposed to concentrations of 100, 316, 1000 and 3160 mg/ml. No growth inhibition was seen up to a concentration of 316 mg/l, distinct and increasing growth inhibition was observed at concentrations of 1000 and 3160 mg/ml. Hence concentrations of 313, 625, 1250, 2500, 5000 and 10000 mg/l were used for the main test.

Study design

Test type:
static
Water media type:
other: The water media type follows the provisions of the method: surface, ground, or waste water to be tested
Limit test:
no
Total exposure duration:
16 h

Test conditions

Hardness:
not applicable
Test temperature:
20°C - 21°C
pH:
Initial pH of all test compound solutions and the control solution: 6.88 - 6.94
Dissolved oxygen:
not applicable
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
Nominal concentrations apply according to study report information. Preparation of the inoculation medium is described accordingly.
Details on test conditions:
Both inoculated and non-inoculated (see above) dilution series as well as the control culture flasks were left at 20°C - 21°C in the dark for 16 ± 1 hours (shaker; 75 - 85 rpm).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
1 824 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
3 520 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
EC100
Effect conc.:
10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
After termination of the test period the extinction of the monochromatic light at 436 nm was measured in a 10-mm cell with samples from the four test substance flasks per concentration and the control flasks. Since turbidity occurred in the dilution series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series were used as photometric blank values for the turbidity of the inoculated dilution series.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The following values were calculated
(i) mean extinction value of the control cultures
(ii) mean extinction values of each concentration of the inoculated dilution series

The mean extinction values of each dilution step were plotted against the logarithm of the values of the concentration of the test substance. The extinction values were measured against the non-inoculated series.

The inhibitory effect on cell multiplication (H) was calculated as follows for each tested concentration:

H = (Bc - Bn)/(Bc - B0)

whereas H = inhibitory effect on cell multiplication, in %
Bc = Biomass in the control at the end of the test
Bn = Biomass of the nth concentration at the end of the test
B0 = Biomass of the control at time t0

From the different series the mean was calculated together with the standard deviation.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
IchthyolR was investigated for its ability to inhibit the cell multiplication of the bacteria species Pseudomonas putida according to DIN 38 412 L-8. Cultures of the bacteria were exposed to concentrations ranging from 313 mg to 10000 mg IchthyolR/l.
Under the present test conditions no inhibition of cell multiplication of the bacteria species Pseudomonas putida was observed at concentrations of 313 and 625 mg Ichthyol/l. At concentrations ranging from 1250 to 10000 mg/l a distinct and concentration-related inhibition of cell multiplication was observed.
The following parameters were determined for Pseudomonas putida:

EC10 (16 h) : 1824 mg Ichthyol/I
EC50 (16 h) : 3520 mg Ichthyol/l
EC100 (16 h) : 10000 mg Ichthyol/l.