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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 03/06/1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
yes
Remarks:
see "Principles of methode if other than guideline"
Principles of method if other than guideline:
Method: other: acute bacteria cell multiplication inhibition test
deviations from guideline DIN 38412, part 8: - duration in test 18h (Guideline: 15 70 17h)
- temperature: 25°C (Guideline: 20-22°C)
-slightly different composition of the solutions
GLP compliance:
yes
Details on test solutions:
228000mg Guanidine Nitrate/L in sterile MilliQ-water (Millipore Corp., Bedford, Mass., USA)
pH adjusted to 7.0 +-0.1 with 0.01M NaOH
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Pseudomonas putida, obtained from RIVM, Bilthoven, The Netherlands
- Method of cultivation: at 25°C
- Preparation of inoculum for exposure: small amounts of bacteria from a 7-day old stock culture inoculated in fluid nutrient medium in erlenmeyer falsks, turbidity measured at 436nm for a 10mm layer, final turbidity adjusted bymeans of sterile saline that dilution 1 + 9 with saline corresponded with extinction value of a Formazin standard supsension TU/F/436nm=10, these preliminary clutures used for inoculation of the test flasks
Test type:
static
Water media type:
freshwater
Total exposure duration:
18 h
Test temperature:
25°C
pH:
7.0
Details on test conditions:
TEST SYSTEM
- Test vessel: 300ml Erlenmeyer flasks stoppered with aluminium caps
- Material, size, headspace, fill volume: glas, 300ml, 100ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 10
- Biomass loading rate: 10ml of prepared bacterial suspension (TU/F/436nm=10) in 80ml diluted test substance solution, 5ml stock solution I, 5ml stock solution II

Reference substance (positive control):
yes
Remarks:
Methanol (Merck no.6008, The Netherlands)
Key result
Duration:
18 h
Dose descriptor:
EC10
Effect conc.:
ca. 7 125 mg/L
Results with reference substance (positive control):
- Relevant effect levels: TT value of methanol was 4938 mg/l

Guanidine hydrochloride test concentration (mg/l)

vs.

mean corrected extinction at 436 nm:

 1.7  .478
 7.0  .490
 27.8  .495
 111.3  .473
 222.7  .486
 445.3  .497
 890.6  .495
 1781.2  .505
 7125.0  .486
 28500  .238
 114000  .057
 456000  -.009
 Mean: 182400  Mean: .025

According to the study authors the EC10 of Guanidine hydrochloride is 88.7 mg/L.

However, at a Guanidine hydrochloride concentration of 111.3 mg/L one of the triplicate values is clearly an outlier (single extinction values: 0.478, 0.504, 0.508, or corrected for background 0.454, 0.480, 0.484) resulting in a slight drop below the 90% threshold line of 0.479 and distorting the outcome.

Higher concentrations up to 7125 mg/L did not lead to a growth reduction below the 90% threshold. Thus, the result has been re-evaluated, and the true EC10 has been estimated to be ca. 7125 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of Guanidine chloride against microorganisms was studied in a cell multiplication inhibition test using Pseudomonas putida as test organism. A toxicity value (TT) of 7125 mg/L was determined.
Executive summary:

In a 18 h acute toxicity study, the cultures of the microorganism Pseudomonas putida were exposed to Guanidine cloride at nominal concentrations of 1.7 - 456000 mg/L under static conditions in accordance with the guideline of Umweltbundesamt (1979) and slightly modified to DEV DIN 38412 part 8 (1991). Bacterial growth was determined by measurement of the turbidity at 436 nm.

The study is in accordance with standard guidelines with some restrictions.

 

Results Synopsis

 

Test organism: Pseudomonas putida

Test Type: static

18-h-EC10:  7125 mg a.i./L                        

Endpoint(s) Effected: toxicity to microorganisms, growth inhibition

Description of key information

The toxicity of Guanidine hydrochloride to microorganisms was monitored in Pseudomonas putida. A 16-h-EC10 of 7125 mg/L was determined.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
7 125 mg/L

Additional information

In an 18 h acute toxicity study, the cultures of the microorganism Pseudomonas putida were exposed to Guanidine hydrochloride at nominal concentrations of 1.7 - 456000 mg/L under static conditions in accordance with the guideline of Umweltbundesamt (1979) and slightly modified to DEV DIN 38412 part 8 (1991). Bacterial growth was determined by measurement of the turbidity at 436 nm. The study is in accordance with standard guidelines with some restrictions. The 18-h-EC10 was 7125 mg/L.

This result is supported by a study conducted with the read-across substance Guanidine nitrate: The toxicity of Guanidine nitrate to microorganisms was studied in an 18-h-cell multiplication inhibition test using Pseudomonas putida as test organism. Bacterial growth inhibtion was determined by measurement of the turbidity of the bacterial culture. The 18-h-EC10 was 831.8

mg/L.

Justification for read-across:

Guanidine hydrochloride and guanidine nitrate dissociate in aqueous media to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the effects of the ions separately. The chloride ion is a naturally occurring essential ion in human beings with well-known metabolism and mechanisms of action as described in standard textbooks on pharmacology and physiology. As well it is found as salt in the Earth´s crust and is dissolved in seawater. Effects of guanidine hydrochloride are expected to be based primarily on the guanidine ion. The physiological processing of the guanidine ion is expected to be independent of the individual source. Therefore read-across from guanidine nitrate for effects of guanidine dissociated from guanidine hydrochloride is considered valid. This strategy is supported by a quite similar toxicological profile of both substances, as shown in acute toxicity, irritation, sensitization and genotoxic studies.

A more detailed justification for read-across is attached in IUCLID chapter 13.