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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This data don't give sufficient experimental details to assess the reliability.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two hundred and thirty perfumery chemicals at concentrations from 1/500 to 1/10000 were tested in vitro against growing cultures of four bacteria. The chemicals included: acetals, acids, aldehydes, esters, ethers, ketones, lactones, and miscellaneous compounds. Among these compounds, gamma-nonalactone and gamma-undecalactone were tested.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
not specified
Details on test solutions:
No data
Test organisms (species):
other: Bacillus subtilis, Escherichia coli and Staphylococcus aureus
Details on inoculum:
- The test organisms used were: Bacillus subtilis ATCC 9524, Escherichia coli ATCC 1129, Staphylococcus aureus Ox-H (penicillin sensitive) and Staphylococcus aureus ATCC 10390 (penicillin resistante).
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
24 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
37°C
pH:
No data
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
1/500, 1/1000, 1/2000, 1/10000
Details on test conditions:
- Dilutions of each chemical were prepared in sterile nutrient broth (Difco) at 1/500, shaken by hand for several minutes and 7 mL dispensed into sterile tubes. Each tube was inoculated with 0.5 mL of a 24h broth culture of test organism. The tubes were incubated at 37°C for 24h and the presence or absence of visible growth observed. Oftentimes the cloudiness of the mixture would obscure visible growth of the bacteria. To overcome this, growth was checked by transferring three loopfuls of the test mixture into tubes of sterile medium without added chemical. The latter tubes were incubated for 24 hours at 37°C. Failure of growth to occur in the subculture was taken as evidence that the organisms had been killed in the original chemical-broth tubes. When this occurred additional concentrations were prepared (1/1000, 1/2000, 1/10000) and tested in a similar manner.
Reference substance (positive control):
not required
Remarks on result:
not determinable
Details on results:
For gamma-undecalactone:
- growth of B. subtilis, E. coli, S. aureus 10390 and S. aureus Ox-H at 1/500.

For gamma-nonalactone:
- growth of B. subtilis, S. aureus 10390 and S. aureus Ox-H at 1/500.
- growth of E. coli at 1/1000 but not at 1/500 (this substance inhibited E. coli at 1/500 test concentration)

For lactones group (benzo dihydro pyrone, decalactone, gamma-hepta lactone, gamma-nonalactone, gamma-octalactone and gamma-undecalactone): Inhibitiory activity is estimated to be 14%.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

None

Validity criteria fulfilled:
not applicable
Conclusions:
The data presented show that no growth inhibition was observed at 1/500 of the test substance for any bacteria tested.
Executive summary:

Two hundred and thirty perfumery chemicals at concentrations from 1/500 to 1/10 000 were tested in vitro against growing cultures of four bacteria (Bacillus subtilis ATCC 9524, Escherichia coli ATCC 1129, Staphylococcus aureus Ox-H (penicillin sensitive) and Staphylococcus aureus ATCC 10390 (penicillin resistante)) at 37°C during 24 hours.

The chemicals included: acetals, acids, aldehydes, esters, ethers, ketones, lactones, and miscellaneous compounds. Among these compounds, gamma-nonalactone and gamma-undecalactone were tested.

The results show that no growth inhibition was observed at 1/500 of gamma-undecalactone for any bacteria tested. For gamma-nonalactone, growth inhibition at 1/500 was observed only at E. coli. Generaly, inhibitiory activity is estimated to be 14% for the lactones group.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This study was not performed according to international or national guideline, without GLP statement.
Qualifier:
no guideline followed
Principles of method if other than guideline:
First, a large number of materials were screened in a standardized petri plate procedure. Second, materials identified as having significant bacteriostatic activity were tested in liquid cultures to determine their minomum inhibitory concentrations (MIC). Third, a soap fragrance was created from the most active frangrance materials and tested in handwashing panel studies to determine its degerming efficacy. In all phases of this study, known antimicrobial chemicals were included as controls so that direct comparison of the efficacy of the fragrance materials vs. these bacteriostats could be made.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
not specified
Details on test solutions:
No data
Test organisms (species):
other: Staphylococcus aureus, Escherichia coli, Candida albicans and a lipophilic diphteroid probably a Corynebacterium species.
Details on inoculum:
The following microorganisms were used: Staphylococcus aureus, Escherichia coli and Candida albicans, obtained from Monmouth Medical Center, Long Branch, New Jersey; a lipophilic diphteroid probably a Corynebacterium species, obtained from the Department of Dermatology, University of Pennsylvania.
Stock cultures were maintained on agar slants composed of tryptone (Difco), 0.3%; yeast extract (Difco), 0.3%; glucose, 0.3%; K2HPO4, 0.1%, and agar, 1% (referred to as TGY agar). Slants for the diphtheroid strain were supplemented with 0.5% Tween 80 (ICI America, Inc.).
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
24 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
37°C
pH:
For the petri plate screening work, pH was adjusted to 5.5 .
For liquid cultures, the pH was not adjusted (pH 7.0).
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Petri plate-paper disc procedure: 20 µL of the 10% test solutions.
Liquid culture procedure (not performed with gamma-undecalactone): selected chemicals were tested initially at 0.1, 1.0 and 10 ppm to determine the approximate range of activity. When no positive materials were found, these were repeated at 10, 100 and 1000 ppm. Materials selected by the petri plate screen were then tested at 100, 500 and 1000 ppm. Those found positive at 100 ppm were repeated at 10, 50 and 100 ppm.
Details on test conditions:
TEST SYSTEM (petri plate-paper disc procedure)
The growth medium was maintained at 42°C during dispensing into plastic petri plates. The medium was seeded at 3.3% with 24 hour shake cultures of the appropriate organisms and dispensed at 8 mL per plate. Paper discs were soaked with 20 µL of the 10% test solutions, and the discs were immediately applied to the center of the solidified seeded agar, one disc per plate. All materials were run in duplicate. It was found that 20µL was uniformly absorbed by the discs without excessive wetting. Total material on the disc was 2 mg for most samples. For those materials available in less than neat form, correspondingly less material was placed on the disc (e.g. a 50% absolute actually had only 1 mg on the disc, the remainder being the solvent used for that material). Control discs with 20 µL of 95% ethanol had no effect on growth of any of the organisms. Plates were incubated in an upright position at 37°C for 18 to 24 hours, during which time an adequate microbial lawn developed.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : zone of inhibition (mm)
Reference substance (positive control):
yes
Remarks:
antimicrobial chemicals (Hexachlorophene, trichlorocarbanilide and trichlorohydroxy diphenyl ether)
Remarks on result:
not determinable
Details on results:
For gamma-undecalactone: No inhibition of growth was detected for S. aureus and E. coli, but the zone of inhibition is 11 mm for C. albicans. The minimum inhibitory concentrations (MIC) were not determined.

For gamma-nonalactone:
- growth of B. subtilis, S. aureus 10390 and S. aureus Ox-H at 1/500.
- growth of E. coli at 1/1000 but not at 1/500 (this substance inhibited E. coli at 1/500 test concentration)

For lactones group (benzo dihydro pyrone, decalactone, gamma-hepta lactone, gamma-nonalactone, gamma-octalactone and gamma-undecalactone): Inhibitiory activity is estimated to be 14%.
Results with reference substance (positive control):
See table 6.1.7/1 in "Any other information on results incl. tables".
Reported statistics and error estimates:
None

Table 6.1.7/1: controls

Controls

S. aureus

E. coli

C. albicans

Diphtheroid

Zone diam. mm

MIC ppm

Zone diam. mm

MIC ppm

Zone diam. mm

MIC ppm

Zone diam. mm

MIC ppm

Hexachlorophene

13

0.10

12

50

0

50

0

100

Trichlorocarbanilide

11

0.08

0

>200

0

100

0

50

Trichlorohydroxy diphenyl ether

25

0.04

22

0.06

12

6.0

0

50

Validity criteria fulfilled:
not applicable
Conclusions:
No inhibition of growth was detected for S. aureus and E. coli, but the zone of inhibition is 11 mm for C. albicans. The minimum inhibitory concentrations (MIC) of the test substance were not determined. In conclusion, the test substance has not antimicrobial activity comparable to well known bacteriostatic agents.
Executive summary:

This study, not according to international or national guideline, was performed to assess the antimicrobial activity of fragrance raw materials. First, a large number of materials were screened in a standardized petri plate procedure (whereof gamma-undecalactone). Second, materials identified as having significant bacteriostatic activity were tested in liquid cultures to determine their minimum inhibitory concentrations, MIC (not performed for gamma-undecalactone). Third, a soap fragrance was created from the most active fragrance materials and tested in handwashing panel studies to determine its degerming efficacy (not performed for gamma-undecalactone). In all phases of this study, known antimicrobial chemicals were included as controls so that direct comparison of the efficacy of the fragrance materials vs. these bacteriostats could be made.

For the preliminary screening (petri plate-paper disc procedure), the growth medium was maintained at 42°C during dispensing into plastic petri plates. The medium was seeded at 3.3% with 24 hour shake cultures of the appropriate organisms and dispensed at 8 mL per plate. Paper discs were soaked with 20 µL of the 10% test solutions, and the discs were immediately applied to the center of the solidified seeded agar, one disc per plate. All materials were run in duplicate. It was found that 20µL was uniformly absorbed by the discs without excessive wetting. Total material on the disc was 2 mg for most samples. For those materials available in less than neat form, correspondingly less material was placed on the disc (e.g. a 50% absolute actually had only 1 mg on the disc, the remainder being the solvent used for that material). Control discs with 20 µL of 95% ethanol had no effect on growth of any of the organisms. Plates were incubated in an upright position at 37°C for 18 to 24 hours, during which time an adequate microbial lawn developed.

For gamma-undecalactone, no inhibition of growth was detected for S. aureus and E. coli, but the zone of inhibition is 11 mm for C. albicans. The minimum inhibitory concentrations (MIC) of the test substance were not determined. In conclusion, this fragrance has not antimicrobial activity comparable to well known bacteriostatic agents.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This study was not performed according to guideline. Little information was provided in this report. No sufficient information was provided to assess the fiability of the results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline followed.
GLP compliance:
no
Specific details on test material used for the study:
- Water solubility (under test conditions): insoluble
- density: 1.0 g/mL
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- stock solution: 2001 mg of test substance in 1000 mL H2O dist (final concentration in the test: 1000.5 mg/L, see table 1 in "Any other information on results incl. tables".
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: activated sludge from a wastewater treatment plant laboratory, with municipal wastewater
- Method of cultivation: no data
- Preparation of inoculum for exposure: concentration of 1 g/L dry matter
- Pretreatment: no data
- Initial biomass concentration: 1g/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
30 min
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
No data
pH:
No data
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
1000.5 mg/L test concentration.
Details on test conditions:
TEST SYSTEM
- No. of vessels per concentration (replicates): 1

TEST MEDIUM / WATER PARAMETERS
deionised water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate

TEST CONCENTRATIONS
- limit test at 1000.5 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
See table 6.1.7/1 in "Any other information on results incl. tables".
- TOC = 755 mg/g
- DOC = 750 mg/g
- ThOC = 706 mg/g
- COD = 2579 mg/g
- BOD = 1539 mg/g
- COD/TOC = 3.4
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 30min-EC50 = 14 mg/L.
Reported statistics and error estimates:
None

Table 6.1.7/1: test procedure and results

Vessel n°

1

Test substance concentration (mg/L)

1000.5

DOC concentration (mg/L)

751

Stock solution (mL/vessel

125

Synthetic medium (mL/vessel)

8

Inoculum (mL/vessel)

50

Dry substance (g/L)

1

Deionised water (mL/vessel)

67

Total volume per vessel (mL)

250

Respiration rate (O2 mg/(L*h))

26

Respiration rate of medium blank (reference value) : 20 mg/(L*h).

Significant effet, if deviation is > +/- 20% from the reference value.

Validity criteria fulfilled:
not specified
Conclusions:
According to the little information provided in this report, the respiration rate of medium blank (reference value) was 20 mg/(L*h). With the test substance, the respiration rate was 26 mg O2/(L*h). Considering that a significant effect is detected if the deviation from the reference value is > +/- 20%, no significant effect is determined. The 30-min EC50 was estimated to be greater than the highest concentration tested 1000.5 mg/L.
Executive summary:

This study, not according to international or national guideline, was performed to assess the toxicity of the test substance (gamma-decalactone) on the respiration of activated sludge Activated Sludge.

A limit test was performed at 1000.5 mg/L with activated sludge from a wastewater treatment plant laboratory with municipal wastewater.

According to the little information provided in this report, the respiration rate of medium blank (reference value) was 20 mg/(L*h). With the test substance, the respiration rate was 26 mg O2/(L*h). Considering that a significant effect is detected if the deviation from the reference value is > +/- 20%, no significant effect is determined. The 30-min EC50 was estimated to be greater than the highest concentration tested 1000.5 mg/L. No sufficient information was provided in this report to assess the fiability of the results.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This study was performed according to German standard guideline DIN 38412, part 27. Little information was provided in this report. No sufficient information was provided to assess the fiability of the results.
Qualifier:
according to
Guideline:
other: DIN 38412, part 27
Deviations:
not specified
Principles of method if other than guideline:
Measurement of oxygen consumption of a bacterial culture.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
yes
Details on test solutions:
No data
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- strain: DSM 50026
- storage: 4°C
- Method of cultivation: the preculture is recorded in 0.9% NaCl solution under sterile conditions.
- Preparation of inoculum for exposure: the harvested cells for 10 min at 6000 r and 10°C centrifuged in a refrigerated centrifuge, washed twice in phosphate buffer washed and resuspended in buffer.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
No data
Test temperature:
22 +/- 2°C
pH:
No data
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 78, 156, 312, 625 and 1250 mg/L
Details on test conditions:
TEST SYSTEM
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
25 g/L Nutrient broth, 18 g/L agar

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : measurement of oxygen consumption by bacteria

TEST CONCENTRATIONS
78, 156, 312, 625 and 1250 mg/L
Reference substance (positive control):
no
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
800 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
See table 6.1.7/1 in "Any other information on results incl. tables".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data

Table 6.1.7/1: Test results

Concentration (mg/L)

Oxygen consumption (mg/L*min)

Average (mg/L*min)

% of control

1250

0/0

0

0

625

1.3/1.3

1.3

79.3

312

1.7/1.7

1.7

103.7

156

1.65/1.65

1.65

100.6

78

1.7/1.7

1.7

103.7

Control

1.65/1.5

1.64

100

Solubilisant

1.7/1.7

Validity criteria fulfilled:
not specified
Conclusions:
According to the little information, the 30min-EC50 was evaluated to be 800 mg/L.
Executive summary:

This study, according to German standard guideline DIN 38412 part 27, was performed to assess the toxicity of the test substance (gamma-decalactone) on microorganisms Pseudomonas putida.

Five test concentrations were prepared with two controls (control and solvent control). Oxygen consumption was recorded after 30 min of exposure.

According to the little information, the 30min-EC50 was evaluated to be 800 mg/L. No sufficient information was provided in this report to assess the fiability of the results.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to the RAAF document in IU section 13 for detailed justification.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Validity criteria fulfilled:
not applicable
Conclusions:
According to the little information provided in this report on the source substance, tthe 30-min EC50 was estimated to be greater than the highest concentration tested 1000.5 mg/L for the target substance.
Executive summary:

An experimental study is available on a source substance. The target and the source substances have similar mechanism of action and share a common basic structure: a linear saturated 4hydroxycarboxylic acid derived-lactone with 10 or 11 carbons, for the source or target substances, respectively.

This study, not according to international or national guideline, was performed to assess the toxicity of the source substance on the respiration of activated sludge Activated Sludge.

A limit test was performed at 1000.5 mg/L with activated sludge from a wastewater treatment plant laboratory with municipal wastewater.

According to the little information provided in this report, the respiration rate of medium blank (reference value) was 20 mg/(L*h). With the test substance, the respiration rate was 26 mg O2/(L*h). Considering that a significant effect is detected if the deviation from the reference value is > +/- 20%, no significant effect is determined. The 30-min EC50 was estimated to be greater than the highest concentration tested 1000.5 mg/L for the source and target substance (by read-across). No sufficient information was provided in this report to assess the fiability of the results.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to the RAAF document in IU section 13 for detailed justification.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
800 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Validity criteria fulfilled:
not applicable
Conclusions:
According to the little information on the source substance, the 30min-EC50 was evaluated to be 800 mg/L for the target substance.
Executive summary:

An experimental study is available on a source substance. The target and the source substance have similar mechanism of action and share a common basic structure: a linear saturated 4hydroxycarboxylic acid derived-lactone with 10 or 11 carbons, for the source or target substances, respectively.

This study, according to German standard guideline DIN 38412 part 27, was performed to assess the toxicity of the source substance on microorganisms Pseudomonas putida.

Five test concentrations were prepared with two controls (control and solvent control). Oxygen consumption was recorded after 30 min of exposure.

According to the little information, the 30min-EC50 was evaluated to be 800 mg/L for the source and target substance (by read-across). No sufficient information was provided in this report to assess the fiability of the results.

Description of key information

Read-Across, German standard guideline DIN 38412 part 27, WoE, validity 4:

30min-EC50 (Pseudomonas putida) = 800 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
800 mg/L

Additional information

A weight of evidence approach was used with four studies. The first and second studies were performed on the registered substance and the third and fourth studies were performed on a similar substance, a linear saturated 4-hydroxycarboxylic acid derived-lactone with 10 carbons (one carbon less in the aliphatic chain than the registered/target substance). Both substances have similar mechanism of action (MechoA 2.1) and share a common basic structure. The source substance is considered adequate for read-across purpose. Please refer to the RAAF document in IU section 13 for detailed justification.

The first study is a publication (Maruzzella and Bramnick, 1961) which evaluates the antibacterial properties of perfumery chemicals. Two hundred and thirty perfumery chemicals at concentrations from 1/500 to 1/10 000 were tested in vitro against growing cultures of four bacteria (Bacillus subtilis ATCC 9524, Escherichia coli ATCC 1129,Staphylococcus aureus Ox-H (penicillin sensitive) and Staphylococcus aureus ATCC 10390 (penicillin resistante)) at 37°C during 24 hours. The chemicals included: acetals, acids, aldehydes, esters, ethers, ketones, lactones, and miscellaneous compounds. Among these compounds, the registered substance was tested. The results show that no growth inhibition was observed at 1/500 of the registered substance for any bacteria tested. Generaly, inhibitiory activity is estimated to be 14% for the lactones group.

The second study is also a publication (Morris et al., 1979) which assess the antimicrobial activity of fragrance raw materials. First, a large number of materials were screened in a standardized petri plate procedure (whereof the registered substance). Second, materials identified as having significant bacteriostatic activity were tested in liquid cultures to determine their minimum inhibitory concentrations, MIC (not performed for the registered substance). Third, a soap fragrance was created from the most active fragrance materials and tested in handwashing panel studies to determine its degerming efficacy (not performed for the registered substance). In all phases of this study, known antimicrobial chemicals were included as controls so that direct comparison of the efficacy of the fragrance materials vs. these bacteriostats could be made. For the preliminary screening, no inhibition of growth was detected for the registered substance on S. aureus and E. coli, but the zone of inhibition is 11 mm for C. albicans. The minimum inhibitory concentrations (MIC) of the test substance were not determined. In conclusion, this fragrance has not antimicrobial activity comparable to well known bacteriostatic agents.

The third study, according to German standard guideline DIN 38412 part 27, was performed to assess the toxicity of the source substance on microorganisms Pseudomonas putida. Five test concentrations were prepared with two controls (control and solvent control) and oxygen consumption was recorded after 30 min of exposure. According to the little information, the 30min-EC50 was evaluated to be 800 mg/L. No sufficient information was provided in this report to assess the fiability of the results.

The fourth study, not according to international or national guideline, was performed to assess the toxicity of the source substance on the respiration of activated sludge. A limit test was performed at 1000.5 mg/L with activated sludge from a wastewater treatment plant laboratory with municipal wastewater. According to the little information provided in this report, no significant effect is determined at the concentration tested. The respiration rates of blank and test substance were 20 and 26 mg O2/(L*h), respectively. The 30-min EC50 was estimated to be greater than the highest concentration tested of 1000.5 mg/L. No sufficient information was provided in this report to assess the fiability of the results.

Based on the first and second studies, not performed according to test guideline, the registered substance was not considered toxic to microorganism and no endpoint values were measured. To have a key value for chemical safety assessment, the experimental studies performed on the source substance were used in a weight of evidence approach to assess the toxicity of the registered/target substance to microorganisms. In these studies, the 30min-EC50 values were determined at 800 mg/L and >1000.5 mg/L for Pseudomonas putida and activated sludge, respectively. The lowest value was considered as the key value for the chemical safety assessment.

The structural similarity, physicochemical and environmental fate properties, and ecotoxicological profile between the target and the source substances are pronounced. As no toxicity was observed on microorganisms for the target substance (considered as the most toxic substance with 11 carbons), it's considered suitable and scientifically justified to read-across the data on the source substance to fill the toxicity to microorganisms endpoint in the present dossier.