Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2011-08-02 - 2012-03-
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Klimisch 1 source record, but performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
not required
Details on sampling:
30 min, 3h (end of the test)
Vehicle:
no
Details on test solutions:
2500 mg of test item was dissolved in water with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 Liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions.
The pH of the test item stock solutions were measured and adjusted to between pH 7 to pH 8.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Inoculum: mixed population of activated sewage sludge microorganism
- Date of collection: 5 March 2012
- Location: aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

PREPARATION OF INOCULUM
The sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.0, measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allow to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period described.
Hardness:
No details available.
Test temperature:
20 +/- 2 °C
pH:
Stock solutions:
25 mg/L: 7.5 (after adjustment)
250 mg/L: 7.4 (no adjustment necessary)
2500 mg/L: 7.8 (after adjustment)
Dissolved oxygen:
10 mg/L: 4.3 mg O2/L (start), 2.2 mg O2/L (end)
100 mg/L: 4.3 mg O2/L (start), 2.2 mg O2/L (end)
1000 mg/L; R1: 4.7 mg O2/L (start), 1.8 mg O2/L (end); R2: 5.4 mg O2/L (start), 1.9 mg O2/L (end), R3: 5.5 mg O2/L (start), 2.1 mg O2/L (end)
Salinity:
Not applicable.
Nominal and measured concentrations:
10, 100 and 1000 mg/L (3 replicates for 1000 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Aeration: clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 L/min
- No. of vessels per concentration (replicates): 1000 mg/L: 3
- No. of vessels per control (replicates): 4

TEST WATER
Deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

OTHER TEST CONDITIONS
- Adjustment of pH: Stock solutions of test item were adjusted to between pH 7 to pH 8.

SYNTHETIC SEWAGE
16 g Peptone, 11 g Meat extract, 3 g Urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 dissolved in 1 L of water with aid of ultrasonication. The pH of the stock was 7.1, measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. The synthetic sewage was added to each test vessel to act as a respiratory substrate.

RANGE FINDING STUDY
No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

PREPARATION OF TEST SYSTEM
16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask. After aeration, the procedure was repeated at 15 min intervals for the second control followed by the reference item vessels and two further control vessels were prepared.
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration was measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
No statistically significant toxic effects were shown at all of the test concentrations employed.
Results with reference substance (positive control):
EC20(3h): 3.0 mg/L
EC50(3h): 8.8 mg/L (95 % CL: 6.9 - 11 mg/L)
EC80(3h): 26 mg/L
Reported statistics and error estimates:
No statistics and error estimates reported.
Validity criteria fulfilled:
yes
Remarks:
Validity criteria of the OECD Guideline were fulfilled.
Conclusions:
The study is regarded as a valid guideline study with certificated GLP compliance. No statistically significant toxic effects were shown at all of the test concentrations employed.
Executive summary:

The toxicity effects of the test substance towards aquatic microorganisms were investigated according to OECD Guideline 209 under certificated GLP compliance (Wadsley, 2012). A mixed population of activated sewage sludge microorganisms, collected at the aeration stage of a predominantly domestic sewage treatment plant, were used as inoculum. This inoculum was exposed to an aqueous solution of the test substance at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 +/- 2 °C with addition of synthetic sewage as respiratory substrate. 3,5 -dichlorophenol was used as reference compound, resulting in an EC50 value of 8.8 mg/L, confirming the sustainability of the test procedure. Furthermore, 4 control replicates were run in parallel. No statistically significant toxic effects were shown at all of the test concentrations employed. Therefore, no further testing was employed and the EC50(3h) was determined to be greater than 1000 mg/L with a corresponding No Observed Effect Concentration (NOEC) of 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of this concentration. The test substance solutions appeared during the test as pale brownish dispersion, no undissolved material was visible. The validation criteria of the applied test guideline have been satisfied.

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1990-02-15 - 1990-03-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non-GLP, performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Bringmann,G., Kuhn,R.: WAF 10 (1977), p. 87-98
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
Medium for stock culture: (Sodium nitrate: 1.060 g, Dipotassium hydrogenphosphate (anhydr): 0.600 g, Potassium dihydrogenphosphate: 0.300 g, Magnesiumsulphate 7H20: 0.200 g, D(+)Glucose: 10.000 g, Agar(0X0ID)No2: 18.000 g, Ferri citrate (19 % Fe): 0.060 g, Trace element solution: 1.500 mL) Dissolved in 1000 mL bidestilled water the above materials were autoclaved at 0.8 bar for 1 hour. To the sterilized solution 3 mL of vitamin solution were added under aseptic conditions. The solution dosed into test tubes for slant agars.
Medium for preculture A: (Sodium nitrate: 1.060 g, dipotassiumhydrogenphosphate (anhydr): 0.600 g, Potassiumdihydrogenphosphate: 0.300 g, Magnesiumsulphate 7H20: 0.200 g, Ferri citrate (19% Fe): 0.060 g, Trace element solution: 1.500 mL) The medium was diluted by 90 mL into 300 mL Erlenmayer flask and autoclaved.
Medium for preculture B: (D-Glucose: 100.00 g, Bidestilled water: 1000.00 mL) The solution was autoclaved and 10.10 mL aliquot were measured
under aseptic condition to solution A right prior to use.
Trace element solution (g/1000 mL solution): (Aluminium sulphate. 18xH20: 0.055 g, Potassium jodide: 0.028 g, Potassium bromide: 0.028 g, Titandioxide: 0.055 g, Stannochloride 2xH20: 0.028 g, Litiumchloride: 0.028 g, Mangandichloride 4xH20: 0.389 g, Boron acid H3B03: 0.614 g, Zinksulphate 7xH20: 0.055 g, Coppersulphate 5xH20: 0.055 g, Nickelsulphate 6xH20: 0.059 g, Cobaltnitrate 6xH20: 0.055 g, Bidistilled water: to 1000.000 mL)
Vitamin solution: (Biotin (D(+)-Biotin): 0.2 mg, Nikotin acid: 2.0 mg, Thiamin HCl: 1.0 mg, p-Aminobensoic acid: 1.0 mg, Panthoten acid (D)- Na salt: 0.5 mg, Pyridoxamin HCl: 5.0 mg, Cyanocobalamin (Vitamin B12): 2.0 mg, Bidistilled water: 100.0 mL).
Test medium I (Stock solution I): (Trace element solution: 30 mL, Nutrient solution: 500 mL, D(+)Glucose solution: 500 mL, D(+)Glucose solution (D(+)Glucose: 20 g, Bidistilled water: 500 mL)
Nutrient solution: (NaN03: 4.24 g, K2HP04: 2.40 g, KH2P04: 1.20 g, Bidistilled water: 500.00 mL): Sterilized 115 °C in autoclave for 30 min. The stock solution I and the nutrient solution were combined under aseptic conditions after they were cooled.
Test medium II (Stock solution II): (Ferrisulphate 7xH20: 0.20 g, Magnesiumsulphate 7xH20: 4.00 g, Bidistilled water: 1000.00 mL). The solution is sterilized by membrane filtration (0.2 µm pore sizes).
Sodium chloride solution: (Sodium chloride: 0.500 g, Bidistilled water: 1000.000 mL). Sterilized 121 °C autoclave for 30 minutes.
Formazine solution for calibration of the instrument: A: (Hexamethylentetramin: 10.0 g). Dissolved in 100 mL bidistilled water; B: (Hydrazinsulphate: 1.0 g). Dissolved in 100 mL bidistilled water. 5 mL of solution A were added to 5 mL of solution B and left stand for 24 h. At room temperature the solution was supplemented with bidistilled water ad 100 mL.
- Method of cultivation: The stock culture of Pseudomonas putida was kept in culture tubes containing slant agar medium.
- Preparation of inoculum for exposure: The preculture was inoculated from the stock culture into the medium for the preculture and incubated for 18 +/-2 h at 25 °C.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Storage of the tester strains: Frozen permanent copies of the tester strains are stored at -80 C. They were prepared from fresh overnight cultures to which DMSO was added as a cryoprotective agent. In addition of frozen permanents we have cultures of the tester strains on master plates which can be stored at 25 °C, for a week. After 1 week a new stock culture is prepared by inoculation and incubation at 25 °C for 24 hours. The culture is kept at this temperature until it is used.
- Laboratory culture: Tester strains cultures were grown in preculturing broth. Cultures were inoculated from master plates and were incubated
for 18 +/-2 h at 25 °C.
- Type of strain used: Pseudomonas putida 173019 OKI (Trevisan 1889) Migula 1895 258 HNCMB (1980) H.Schütze, London NCTC 912 urine 1921
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
18 h
Remarks on exposure duration:
+/- 2 h
Post exposure observation period:
No details are reported.
Hardness:
No details given.
Test temperature:
25 °C
pH:
No details given.
Dissolved oxygen:
No details given.
Salinity:
No details given.
Nominal and measured concentrations:
0.01, 0.02, 0.03, 0.04, 0.06, 0.09, 0.13, 0.20, 0.30, 0.44 and 0.67 g/mL
Details on test conditions:
The extinction of the bacterium suspension was determined at 436 nm in cuvette of 10 mm light path. By diluting the suspension the extinction was set to the value measured for formazine standard. From the stock solution of the substance 4 series of 11 grade dilution was prepared at log 1.5 intervals. Each test flask contained 44 mL diluted test solution. 3 series of test flask the followings were added: A) Stock solution 1: 2.75 mL, B) Stock solution II: 2.75 mL, C) Bacterium suspension: 5.50 mL. The 4-th series of flasks received the same solutions but the bacterial suspension was replaced by bidistilled water. For preparing a test substance-free control culture a flask was filled with the above solutions and bacterial suspension and with distilled water substituting the test substance solution.
Reference substance (positive control):
yes
Remarks:
control medium
Duration:
18 h
Dose descriptor:
other: MIC (Minimum Inhibitory Concentration)
Effect conc.:
0.12 other: g/mL
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of nitrification rate
Remarks on result:
other: (In 0.12 = -2.11)
Details on results:
No further details are reported.
Results with reference substance (positive control):
No details are reported.
Reported statistics and error estimates:
No details are reported.
Validity criteria fulfilled:
yes
Remarks:
Valid guideline study.
Conclusions:
The study is regarded as a valid guideline study. Unfortunately the result (MIC value) can neither be used for the chemical safety assessment nor for the risk assessment.
Executive summary:

The acute toxicity to microorganisms of the test substance was tested with Pseudomonas putida according to Bringmann and Kühn (Oláh and Kiss, 1990). In this experiment the bacterial growth is quantitated by turbidimetry i.e. by measuring the extinction of both the test and the control medium. Concentrations of the test substance were 0.01, 0.02, 0.03, 0.04, 0.06, 0.09, 0.13, 0.20, 0.30, 0.44 and 0.67 g/mL. The stock culture of P. putida was kept in culture tubes containing slant agar medium. The preculture was inoculated from the stock culture and incubated for 18 +/- 2 hours at a temperature of 25 °C. The extinction of the bacterium suspension was determined at 436 nm in a cuvette of 10 mm light path with a U-3200 type HITACHI spectrometer. By diluting the suspension the extinction was set to the value measured for formazine standard. A control culture was prepared with the same solutions (stock solution I and II together with bacterial suspension) but destilled water was substituting the test substance solution. As result a MIC (Minimum Inhibitory Concentration) of 0.12 g/mL was determined out of the extinction values using a graphic method.

Description of key information

Toxicity to microorganisms: EC50(3h) > 1000 mg/l, NOEC(3h) = 1000 mg/l, based on respiration inhibition, for a mixed population of activated sewage sludge microorganism (read-across from PPS, OECD 209, static, GLP)

Toxicity to microorganisms: Minimum inhibitory concentration = 120 g/l for Pseudomonas putida (read-across from PPS, method acc. Bringmann and Kühn, static)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information