Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
toxicity to terrestrial arthropods: long-term
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference

Overall, considering all the above information together, the test substance is considered to be readily biodegradable undergoing complete mineralization.

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Study 1:A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the test substance, C16-18 and C18-unsatd. TMAC (96% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the test substance was evaluated using detoxification methods through the addition of the sorbents silica gel and humic acid at two different concentrations. Activated sludge or river water was used as inoculum in the Closed Bottle test. In addition, a sorbent free test group without any deviations from the guideline was included as a ‘negative control’, to demonstrate the toxicity of the test substance and to demonstrate the positive detoxifying effects of the sorbents. Ammonium chloride was omitted from the medium to prevent nitrification for all groups except the sorbent free group. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the test substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the test substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The theoretical oxygen demand (ThOD) of test substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still >60% biodegradation was achieved within 28 days using activated sludge as inoculum and 1 g silica gel / bottle for detoxification. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption and this was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial test substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the test substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance was considered further for the main study (Geerts, 2020).

The main study was conducted to determine the ready biodegradability of the test substance, C16-18 and C18-unsatd. TMAC (96% active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the domestic wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. The measured dry weight of the inoculum was 3.2 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.9°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of biochemical oxygen demand (BOD) bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the test substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.86 and 3.06 g oxygen/g active ingredient, respectively. According to the results of this study, the test substance did not cause a reduction in the endogenous respiration at Day 7. The test substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The test substance was biodegraded by 67% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the test substance and using a correction for the oxygen consumption by the nitrification, the test substance was biodegraded by 63% at day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at Day 28. Furthermore, the differences of the replicate values at Day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 75%. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period Under the study conditions, the test substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020).

Study 2:A study was conducted to determine the ready biodegradability of the test substance, C16-18 and C18-unsatd. TMAC (49% active in isopropanol and water), in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test substance at 2 mg/L was incubated with inoculums from river water and O2 consumption was followed over a period of 28 days. The test was performed using 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing river water, silica gel and test substance, and 6 bottles containing sodium acetate and river water. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6. 7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of 0.3 L BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. The test was found to be valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 71% and 69% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. Therefore, the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration in presence of silica gel, hence was considered to be non-inhibitory to the inoculum. Under the conditions of the study, the test substance is considered to be readily biodegradable (van Ginkel CG, 2010).

Study 3:A study was conducted to determine the biodegradation in water of the read across substance, C18 TMAC (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the read across substance. The concentrations of the read across substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the read across substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The read across substance was biodegraded by 77% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the read across substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case – as longer chains tend to biodegrade more slowly than shorter chains, the test substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the read across substance.

The use of silica gel in the key study on biodegradation is supported by the findings from van Ginkel 2008, which showed that silica gel was the best adsorbent as compared to lignosulphonic acid and humic acid (see Figure 1 in CSR):

Overall, the results obtained with the test substance are in agreement with what is reported in the literature, as summarized below in Table 4.4.

Table 4.4. Compilation of ready biodegradability test results obtained with quaternary ammonium salts (adapted van Ginkel, 2007)

Substance

Test

Results at Day 28 (%)

Hexadecyltrimethylammonium

Chloride (C16 TMAC)

Headspace Carbon

Dioxide

75*

Octadecyltrimethylammonium

Chloride (C18 TMAC)

Sturm test

>70

Cocotrimethylammonium (Coco TMAC)

Closed bottle

>60

Octylbenzyldimethylammonium chloride (C18 ADBAC)

MITI

>80

Tetradecylbenzyldimethylammonium

Chloride (C14 ADBAC)

MITI

>80

Decylbenzyldimethylammonium

Chloride (C10 ADBAC)

Closed bottle

>60

*Mean from 10 laboratories; also cited in OECD TG 310 (adopted on 23 March 2006)

In addition, several literature data are available to clarify the metabolic basis of degradation by micro-organisms. Bacteria identified as Pseudomonas sp capable of degrading alkyltrimethylammonium salts were isolated from activated sludge (van Ginkelet al., 1992; Takenakaet al., 2007). Alkyltrimethylammonium salts with octadecyl, hexadecyl, tetradecyl, dodecyl, decyl, octyl, hexyl and coco alkyl chains supported growth of the isolates, showing the broad substrate specificity with respect to the alkyl chain length. Alkanals, and fatty acids can also serve as a carbon and energy source (van Ginkelet al., 1992; Takenakaet al., 2007). In simultaneous adaptation studies,1H nuclear magnetic resonance spectrometry (1H-NMR) and GC-MS showed that acetate, alkanals and alkanoates are the main intermediates of alkyltrimethylammmonium salt degradation, indicating that the long alkyl chain is utilized for microbial growth (van Ginkelet al., 1992; Nishiyama and Nishihara, 2002; Takenakaet al., 2007). Trimethylamine is stoichiometrically produced by pure cultures of microorganisms growing with the alkyl chain of alkyltrimethylammonium chloride as the sole source of carbon. The cleavage of the C-alkyl-N bond of alkyltrimethylammonium salts resulting in the formation of trimethylamine is initiated by a mono-oxygenase (van Ginkelet al., 1992). Additional evidence of the cleavage of the C-alkyl-N bond as the initial degradation step of alkyltrimethylammonium salts was presented by Nishiyamaet al. (1995) and Takenakaet al. (2007).

Dehydrogenase activity present in cell-free extract of hexadecyltrimethylammonium chloride-grown cells catalysed the oxidation of alkanal to fatty acids. The route of the fatty acid degradation is by β-oxidation. Trimethylamine, a naturally occurring compound is readily biodegradable (Pitter and Chudoba 1990). Complete degradation of trimethylamine is demonstrated through the assessment of the biodegradation pathway. Trimethylamine is degraded by methylotrophic bacteria through successive cleavage of the methyl groups (Large, 1971; Meiberg and Harder, 1978). Consortia of microorganisms degrading the alkyl chain of alkyltrimethylammonium salts and trimethylamine are therefore capable of complete (ultimate) degradation of alkyltrimethylammonium salts. Complete degradation of alkyltrimethylammonium salts using a mixed culture has been demonstrated by Nishiyamaet al. (1995). More recently, Nishiyama and Nishihara (2002) have isolated aPseudomonas spcapable of degrading both the alkyl chain and trimethylamine.  Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkyltrimethylammonium salts results in the formation of water, carbon dioxide and ammonium (see Figure 2 in the CSR).

Further, according to the evidence presently available on the biodegradation rate, microorganisms readily oxidize the hydrophobic alkyl chains of the cationic surfactants, which is followed by a slower oxidation of the hydrophilic moiety (the corresponding amines) (van Ginkel, 2004). The above biodegradation process for the two moieties plays a key role in the differences in the results between the different cationic surfactants. However, based on the available experimental data and literature evidence, the alkyl chains and the trimethylamine of the test substance is readily biodegradable.

Overall, considering all the above information together, the test substance is considered to be readily biodegradable undergoing complete mineralization.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

Please note:A new better quality biodegradation study is available under the biocides consortium for C12-16 ADBAC substance. LoA discussions are ongoing with the data owners and it is expected to be completed in next few weeks. Another dossier update will be submitted as soon as this study is made available. A brief summary of the data has been shared by the data owner (see below) to allow proceeding with risk assessment.

Based on the results of the read across study, the transformation of the C12 carbon chain of the test substance can be considered to be rapid with DT50 values ranging from 2.2-8.7 days with the SFO model and 1.6 – 7.2 days with the FOMC model.

Half-life in soil:
8.7 d
at the temperature of:
20 °C

Study 1: To ADD THE NEW SOIL BIODEGRADATION STUDY

Study 2:A study was conducted to determine the aerobic biodegradation of the read across substance, C12-16 ADBAC (50% active in water) in loamy soil, according to the US FDA Environmental Assessment Handbook, Technical Assistance Document 3.12 (1987). The study comprised two treatments: test and chemical blank control group, each with three replicates. The read across substance was added into biometers at a concentration of 10 mg carbon per 50 g soil using appropriate amount of deionised water required for bringing the soils to 50-70% of the moisture capacity. Loam was added to the biometers after the test solutions to facilitate uniform moistening of the soils by capillary action. The test was then incubated at 22 ± 3°C and run for approximately 90 d. The side tube of the biometer contained 20 mL 0.2 M KOH for absorbing carbon dioxide produced by the microorganisms. The theoretical CO2 production of the read across substance was calculated from its carbon content. The amounts of carbon dioxide were calculated by subtracting the mean carbon dioxide production in the test systems containing the read across substance and the mean carbon dioxide production level in the control blank. Biodegradation was calculated as the ratio of experimental carbon dioxide production to theoretical carbon dioxide production [ThCO2P]. Under the study conditions, there was 64% degradation of the read across substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days (Ginkel, 1994). Based on the results of the read across study, similar degradation potential and half-life is considered for the test substance.  ​

Based on the most recent and radiolabelled read across study in soil, the transformation of the C12 carbon chain of the test substance can be considered to be rapid with DT50 values ranging from 2.2-8.7 days with the SFO model and 1.6 – 7.2 days with the FOMC model.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

The results of the read across study, supported with the estimated BCF value for the test substance together with its ionic nature indicates a low bioaccumulation potential. The higher experimental BCF value of 79 L/kg ww from the read across study has been considered further for hazard/risk assessment

BCF (aquatic species):
79 L/kg ww

Study 1: A study was conducted to determine the aquatic bioaccumulation of the read across substance, C12 -16 ADBAC (30.64% active; 98.9% radiolabeled purity) in Lepomis macrochirus (bluegill fish) under flow-through conditions, according to EPA OPP 165-4, in compliance with GLP. The blue gill fish were continuously exposed to a nominal concentration of 0.050 mg/L of the read across substance (equivalent to a measured concentration of 0.076 mg/L) in well water for 35 days, followed by transfer of 35 fish into flowing uncontaminated water for a 21-d depuration period. Sampling was carried out on Days 0, 1, 3, 7, 9, 10, 14, 21, 23, 28 and 35 for the exposure period and Days 1, 3, 7, 10, 14 and 21 for the depuration period. Water samples were collected on Day 8 of the exposure period and Day 16 of the depuration for analytic determination of the read across substance concentration. Radiometric analyses of the water and selected fish tissues revealed that the mean steady state bioconcentration factor (BCF) in the edible, non-edible and whole-body fish tissue during the 35 days of exposure to be 33, 160 and 79 L/kg. The half-life for non-edible tissue was attained between Days 14 and 21, while it could not be reached for the edible and whole-body fish tissues by the end of 21-d depuration period. By Day 21 of the depuration period, the 14C residues present on the last day of exposure in the edible, non-edible and whole-body fish tissues had been eliminated by 29, 60 and 44% respectively. Analysis of skin tissue after 35 d of exposure showed residue levels somewhat higher than those observed for edible tissue at the same sampling period, indicating that there is likely significant binding of 14C-ADBAC to the skins and scales of exposed bluegill, as expected behaviour of cationic surfactants. Under the conditions of the study, the whole body BCF of the read across substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989).

Study 2: A study was conducted to determine the tissue distribution of two cationic surfactants mixtures in Rainbow Trout (Oncorhynchus mykiss) following exposure via water for seven days and analysis of different fish tissues. The test chemicals were grouped into two mixtures of six containing 10 alkyl amines and 2 quaternary alkylammonium surfactants: C10 TMAB (as part of MIX 2) and C14 TMAC (as part of MIX 1). Studying chemical mixtures has the advantage that differences in behavior between chemicals are not obscured by biological variability or experimental variables. Bioconcentration studies with mixtures have been shown to provide similar results to studies with single chemicals. The experiments were conducted in 300 L fiberglass aquaria with a water renewal rate of 1.3 L min−1 (MIX 1) and 1.45 L min−1 (MIX 2). A solution of the test chemical mixture in methanol was infused continuously (3.5 and 3.8 μL min−1 for MIX 1 and MIX 2, respectively) into the water inflow using a syringe pump. The intended concentrations of C10 TMAB and C14 TMAC were 59 and 1.3 μg/L (measured). The water temperature was 10 °C and the pH 7.5. The water hardness was estimated to be 1.1 mM Ca2+. For each mixture, the syringe pump was started in an aquarium containing no fish. After 16 h, to allow the concentrations to stabilize, 12 rainbow trout were added. After 7 d of exposure, the fish in the exposure aquaria as well as several unexposed (control) fish were sacrificed followed by blood collection.The surface of the fish posterior of the gills was rinsed with 100% methanol to remove read across substance residues adsorbed to the outer surface of the skin and absorbed in the skin mucus.The fish were then dissected and the liver, the kidney, the gills, and the remaining contents of the abdominal cavity were taken and weighed. Skin and muscle samples were prepared from the upper dorsal region on semi-frozen fish after the methanol rinse had removed the mucus. For 6 fish from each aquarium and 3 control fish, samples of muscle, skin, liver, and gills were homogenized in a bullet blender (muscle and liver) or in a cryo-mill (skin and gill). A sub-sample of 0.5−1.2 g of the homogenate was extracted twice in methanol, employing centrifugation at 4000 rpm for phase separation. Isotope labeled standards of C10 TMAB and C14 TMAC were added to a portion of the extract corresponding to 12−75 mg of the sample. Whole blood was analyzed rather than plasma because of the small quantity of sample available and the anticipated low concentrations. The test chemical concentrations generally increased in the order muscle <blood < skin < gills < liver. Because the mass of extracted mucus was not determined, the concentrations in mucus were normalized to the estimated fish’s total surface area excluding the head, which was not rinsed. The concentration in mucus was on average 3.9 (range 0.9−11.6) times lower than the surface area-normalized concentration in gills. To calculate the quantity of the test chemical in the different tissues, the amount of each tissue in the fish was estimated and multiplied by the concentration in that tissue. The test chemical quantities in the different tissues were then summed to give the body burden in each fish. The apparent BCFs (BCFapp) values at the end of the 7-day exposure were calculated by dividing the surfactant body burden (blood, muscles, liver, gills, skin, mucus) by the fish mass, and dividing this by the average measured concentration in water samples taken during the exposure phase. Under the study conditions, the BCFapp for the two quaternary substances C10 TMAB and C14 TMAC were determined to be 0.1 and 31 L/kg ww, respectively. Mucus, skin, gills, liver, and muscle each contributed at least 10% of body burden for the majority of the test chemicals. In contrast to the analogue alkylamine bases, the permanently charged quaternary ammonium compounds accumulated mostly in the gills and were nearly absent in internal tissues, indicating that systemic uptake of the charged form of cationic surfactants is very slow (Kierkegaard, 2020).

Study 3:The Bioconcentration factor (BCF) value of test substance, C16-18 and C18-unsatd TMAC was predicted using regression-based and Arnot-Gobas BAF-BCF models of BCFBAF v3.02 program (EPI SuiteTMv4.11). The Arnot-Gobas method, takes into account mitigating factors, like growth dilution and metabolic biotransformations, therefore the BCF values using this method is considered to be more realistic or accurate. Therefore, except for ionic, pigments and dyes, perfluorinated substances, for which it is not recommended (as of now), the Arnot-Gobas method is used preferentially used for BCF predictions. Considering that the test substance is an UVCB containing majorly ionic (e.g., (e.g., the quaternary ammonium salts) and few non-ionic constituents (e.g., amines), the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter. The BCF values for the constituents ranged from 3.16 to 162.4 L/kg ww (log BCF: 0.50 to 2.21), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or maximum number of correction factor instances domain criteria as defined in the BCFBAF user guide of EPISuite. Further, given that the major constituents are structurally very similar and vary only in the carbon chain length, a weighted average value, which takes into account the percentage of the constituent in the substance, has been considered to dampen the errors in predictions (if any). Therefore, the weighted average BCF value was calculated as 70.29 L/Kg ww (Log BCF = 1.85). Overall, considering either the individual BCF predictions for the constituents or the weighted average values, the test substance is expected to have a low bioaccumulation potential. However, taking into consideration the model’s training set and validation set statistics and the fact that the training set only contains 61 ionic compounds, the BCF predictions for the individual constituents are considered to be reliable with moderate confidence.

This is further supported by the no bioaccumulation potential evidence observed in in the two toxicokinetic studies in mammals with the read across substance, C12 -16 ADBAC (Selim, 1987 and Appelqvist, 2006). .

Also, the biocides assessment reports available from RMS Italy on Coco TMAC and C12-16 ADBAC, concluded the substances to show low potential for bioaccumulation, based on the results from the above study (Fackler, 1989) and an additional read across to DDAC for the Coco TMAC's assessment ((ECHA biocides assessment report, 2015, 2016). The report concluded the following in the Coco TMAC assessment report:Coco alkyltrimethylammonium chloride is readily biodegradable, is rapidly excreted and does not accumulate in mammals, and it adsorbs onto the fish surface where its irritating action is expressed (therefore accumulation is more related to the concentration of the administered solution). Based on these properties’ bioaccumulation is not expected to be of concern for ATMAC/TMAC. An experimental BCFwhole body of 81 L/kg was determined in a flow-through test with Lepomis machrochirus and the read across substance DDAC (Lonza Cologne GmbH and Akzo Nobel Surface Chemistry AB, same study). A very similar result was obtained for the other quaternary ammonium compound benzyl-C12-16-alkyldimethyl ammonium chloride (C12-16-BKC/ADBAC) in a fish bioconcentration test, which gave a BCFwhole body = 79 L/kg (Akzo Nobel Surface Chemistry AB, access to Lonza Cologne GmbH study). Being both studies equally reliable, the BCFwhole body = 81 L/kg is chosen because related to the lead read across substance (DDAC) and it is slightly higher than the C12-16 BKC/ADBAC endpoint.”

Overall, the results of the read across study, supported with the estimated BCF value for the test substance together with its ionic nature indicates a low bioaccumulation potential. The higher experimental BCF value of 79 L/kg ww from the read across study has been considered further for hazard/risk assessment.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

In line with the C12 -16 ADBAC biocides assessment report and based on the results of the read across study, the 16-d EC50 value of 277 mg a.i./kg dw of soil obtained for Brassica alba (mustard) due to effects on growth has been considered further for hazard/risk assessment.

Short-term EC50 or LC50 for terrestrial plants:
277 mg/kg soil dw

Study 1:

A study was conducted to determine the long-term toxicity of the read across substance, C12-16 ADBAC (49.9% active in water) to terrestrial plants, according to OECD Guideline 208, in compliance with GLP. Three plant species: Sinapis alba (mustard), Trifolium pratense (red clover) and Triticum aestivum (wheat) were used. Using 0.5 L capacity plastic pots, the read across substance was first applied to natural soil at nominal concentrations of 0, 476.6, 856.2, 1540.9, 2772.2 and 4990.0 mg a.i./kg and to sand at nominal concentrations of 0, 28.8, 55.8, 93.4, 166.8 and 300.5 mg a.i./kg. This was followed by planting of 40 seeds per replicate of the three plant species. Analytical verification was performed for the read across substance. Three parameters: emergence, dry and wet weight of the plants were observed. Emergence was recorded daily until stabilisation. The plants in natural soil and sand were harvested 16 and 14 d respectively after 50% of the control seeds had been emerged. Wet and dry weight were determined immediately after harvesting. The test was considered as valid on the basis of percent emergence and further growth of the plant in the water control. The extraction of the active substance proved that the natural soil had a strong sorbing effect and the total recovery was not achieved even when acidified methanol was used as an extraction solvent. That was not the case with quartz sand. The EC50 values in natural soil, based on the effect on emergence and growth were 342, 309, 684 mg a.i./kg ww of soil (or 537, 634 and 1960 mg a.i./kg dw of soil) for S. alba, T. pratense and T. aestivum, respectively; while those in sand were 31, 19, 105 mg a.i./kg ww (or 73, 74 and 141 mg a.i./kg dw) of sand respectively. The difference in toxicity in the two substrates were correlated with the lower bioavailability of test substance in soil due to a stronger adsorption potential. Further, as the toxicity to terrestrial plants in sand is not representative of the natural environment, the EC50 in natural soil was considered as a reasonable worst case for representing toxicity terrestrial plant species. Under the conditions of the study, the 16-d EC50 values in natural soil, based on the effect on emergence and growth were determined to be 342, 309, 684 mg a.i./kg ww or 537, 634 and 1960 mg a.i./kg dw of soil for S. alba, T. pratense and T. aestivum, respectively (Servajean, 2004).

Study 2:

A study was conducted to determine the long-term toxicity of the read across substance, C12-16 ADBAC (49.5% active in water) to terrestrial plants, according to OECD Guideline 208, in compliance with GLP. Three plant species: Phaeolus aureus (mung beans) Brassica alba (mustard) and Triticum aestivum (wheat) were used. Each plant species was sown into treated soil and assessed for 14 - 16 days following germination. For each species, groups of 40 seeds (eight replicate pots of five seeds) were sown into a garden loam soil treated with the read across substance. Untreated controls were also included. Treatment levels for the definitive study were based on the results of a preliminary range finding study. The dose levels of the read across substance used were 156, 313, 625, 1250 and 2500 mg a.i./kg dry soil for mung beans and 12, 37, 117, 375 and 1200 mg a.i./kg dry soil for mustard and wheat. After application and sowing, the pots were checked daily and the number of seedlings emerging recorded. Survival and sub-lethal effects were recorded every day following emergence. Plants were harvested 14-16 days after germination and the wet weights were measured. The plants were then dried before being re-weighed to obtain a dry weight measurement. There was no treatment-related effect on the germination and seedling survival of any of the plant species treated with the read across substance up to the highest tested concentrations. The growth inhibition occurred at higher rates of application for all the plant species. For mung bean, there was 25-40 and 50-75% inhibition at 1250 and 2500 mg a.i./kg, respectively. For mustard, there was 75-80 and >80% inhibition at 375 and 1200 mg a.i./kg, respectively and 50-75% for wheat at 1200 mg a.i./kg. Darker pigmentation was observed for all species at the higher rates of application. Under the conditions of the study the 14-16 d EC50 values based on growth inhibition in mung beans, mustard and wheat were determined to be at 1900, 277 and 670 mg a.i./Kg dry soil respectively (Gray, 2004).

Based on the above studies, same effect levels and low toxicity potential were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further stated that: “The great deviation in the effects recorded in sand

and natural soil can be attributed to the lower bioavailability of C12-16 ADBACin natural soil caused by stronger adsorption to the soil particles as consequence of several binding processes. Since the results obtained in the test with silica sand are considered unrealistic worst case, only data from the tests conducted with natural soils are taken into account (this approach was agreed at TMII2013); among these, the most sensitive species was Brassica alba with an EC50 = 277 mg/kg dw soil (US ISC), which is the endpoint to be taken into account at product authorization stage (ECHA biocides assessment report, 2015). Similar conclusions were drawn in the Coco TMAC biocides assessment report, 2016, where the endpoint was mainly assessed based on read across to DDAC apart from the EQC owned supporting study on C12-16 ADBAC. The lowest EC50 for the most sensitive plant among all the tested species, i.e., EC50 (wet weight growth) = 148 mg/kg dw soil for T. pretense exposed to DDAC and corrected for MW as EC50 = 111.0 mg a.s./kg dw (98.3 mg a.s./kg ww) was selected for risk assessment (ECHA biocides assessment report, 2016).

 

In line with the C12 -16 ADBAC biocides assessment report and given that the read across to C12-16 ADBAC can be justified for the test substance based on a category approach, the 16-d EC50 value of 277 mg a.i./kg dw of soil obtained for Brassica alba (mustard) due to effects on growth has been considered further for hazard/risk assessment.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

Based on the results of the read across study, the 14 d LC50 and NOEC values for the test substance is considered to be at 7070 and 517 mg a.i./kg soil dw.  

Short-term EC50 or LC50 for soil macroorganisms:
7 070 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
517 mg/kg soil dw

Study 1. A study was conducted to determine the toxicity to soil macroorganisms of the read across substance C12-16 ADBAC (49.5% active) according to OECD Guideline 207, in compliance with GLP. Six groups of forty earthworm (Eisenia foetida) were allocated to an artificial soil containing 0, 953, 1715, 3086, 5556 or 10000 mg a.i./kg soil dw (nominal concentrations). No analytical dose verification was performed. Mortality was recorded on Days 7 and 14. Worms were weighed at the beginning and end of the study. After 7 days, all worms at 10000 and 2 worms at 5556 mg a.i./kg soil dw were dead. By Day 14, one additional worm died at 5556 mg a.i./kg soil dw. A treatment-related reduction in body weight was observed. Group mean body weights were affected by treatment with read across substance at 1715 mg a.i./kg soil dw and above. Under the study conditions, the 7 and 14 d LC50 values were 7160 and 7070 mg a.i./kg soil dw, respectively and the NOEC was 953 mg a.i./kg soil dw (nominal) (Rodgers, 2004).

Study 2. A study was conducted to determine the toxicity to soil macroorganisms of the read across substance, C12 -16 ADBAC (51.7% active) according to OECD Guideline 207, in compliance with GLP. Earthworms (Eisenia foetida) were exposed to a single dose of the read across substance at nominal concentrations of 100, 180, 320, 580 or 1,000 mg/kg dw of artificial soil. No analytical dose verification was performed. The individual live weights of the worms were reported after 14 d of exposure. Other effects (pathological symptoms, behaviour of the worms) were reported after 7 and 14 d of exposure. Results of the reference test with 2 -chloracetamide show that the method was sensitive and valid. The substance did not cause a change in behaviour, weight and mortality of the earthworm at any of the tested concentrations after 14 d of exposure. This was probably due to adsorption onto soil. The highest tested concentration without mortality and any other effects was 1000 mg/kg dw. Under the study conditions, the 14 d NOEC in earthworm was 1000 mg/kg dw (or 517 mg a.i./kg dw) and the 14 d LC0 was > 1000 mg/kg dw (or > 517 mg a.i./kg dw) (Noack, 1999).

Based on the above two studies, same effect levels were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further stated that: “The findings of the two tests, although different in absolute values, are not in contrast. Since the second test provides a “higher than” value corresponding to a complete lack of lethal or sublethal effects, the 14d LC50 = 7070 mg/kg dry soil (US ISC) is selected to express the acute toxicity of Alkyl (C12-16) dimethylbenzyl ammonium chloride to soil dwelling invertebrates.” While the biocides assessment report on Coco TMAC, assessed the acute toxicity to earthworms based on a study with the substance itself as well as supporting read across studies from DDAC and C12 -16 ADBAC. All three studies indicated low acute toxicity potential in earthworms. The 14d LC50 = 3260 mg a.s./kg dry soil based on the study with Coco TMAC was selected further for risk assessment.

 

Given that the read across to C12-16 ADBAC can be justified for the test substance based on a category approach, the 14 d LC50 and NOEC values of 7070 and 517 mg a.i./kg soil dw has been considered further for hazard/risk assessment.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference

In line with the C12 -16 biocides assessment report and based on the results of the read across study, the lower 28d EC50 = 153 mg a.i./kg dw and a 28d EC10 = 83 mg a.i./kg dw soildue to inhibition of microorganisms has been considered further for hazard/risk assessment.

Short-term EC50 for soil microorganisms:
153 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
83 mg/kg soil dw

Study 1. A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49.9% active in water) to soil microorganisms, according to OECD Guideline 216, in compliance with GLP. In this study, the inhibition of microbial nitrogen transformation was investigated in sandy loam soil by evaluating the nitrite, nitrate and ammonium formation following 28 d exposure to the read across substance. A volume of 6.04 mL of deionized water containing the read across substance was added to 50-g of soil. The samples were incubated for 7 d at 20°C and at 10% of its water holding capacity. The samples were dosed with read across substance at nominal concentrations 0, 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg soil ww. Analytical dose verification of the stock solutions indicated good correlation with the nominal concentrations. Therefore, doses were presented as nominal concentrations. The nitrogen transformation measurements were carried out at the beginning of the test and at the end at Day 28. The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i./kg soil ww. The EC50 calculated was 130 mg a.i./kg soil ww with 95% confidence limits of 80 and 190 mg a.i./kg soil ww. The EC10, EC20 and EC80 of the read across substance were determined at 70, 90 and 200 mg a.i./kg soil ww respectively. In soil not only formation of nitrate occurs but also reduction of nitrate to nitrogen gas by denitrifying microorganisms. Decrease of the nitrate concentrations in the soil was observed at 400 mg a.s./kg soil ww and higher after 28 d. This was probably the result of the activity of these denitrifying microorganisms. The denitrifying microorganisms were inhibited at 6400 mg a.i./kg soil ww, as only a limited amount of the nitrate was removed after 28 d at this concentration. Under the study conditions, the 28 d EC50 and EC10 values were determined to be at 130 and 70 mg a.i./kg soil ww (i.e., equivalent to 153 and 83 mg a.i./kg soil dw) respectively (van Ginkel, 2004).

Study 2. A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49-51% active in water) to soil microorganisms, according to OECD Guideline 216 and 217, and US EPA OPPTS 850.5100, in compliance with GLP. In this study, the effects of the read across substance on carbon mineralization and nitrogen transformation activity of soil micro-organisms were investigated in two soil types (sandy loam soil and a low humic content sand) by evaluating nitrite, nitrate, ammonium and carbon dioxide formation following 28 d exposure. Fifty grams dry weight of soil samples were mixed with lucerne meal (13:1 carbon:nitrogen) and placed in 100 mL bottles. The samples were incubated in the dark at 20±2°C for 28 d. The moisture content of the samples was checked weekly. The samples were dosed with read across substance at nominal concentrations 0, 10, 100 and 1000 µg a.i./g soil dw. No analytical dose verification was performed for the read across substance. Samples were taken to determine nitrogen metabolite content on days 5 and 28 and the CO2 evolution was determined on Days 5 – 8 and 25 – 28. No significant reduction in ammonium formation was observed. The difference in the CO2 production and nitrogen transformation between the treated and untreated soil samples did not exceed 25% after 28 d of incubation. The highest inhibition recorded was 82.5% in the nitrite formation rate after 5 d at 10 mg a.i./kg soil dw in the sandy loam soil. After 28 d of incubation, however, no relevant effect was observed (<25% reduction). Therefore, it was not necessary to extend the test beyond 28 d. Under the conditions of the study, the read across substance was therefore considered to have a low potential for adversely affecting the microbial functions of sandy loam and low humic content sand soils and the 28 d EC50 and NOEC were considered to be at >1000 and ≥1000 µg a.i./g soil dw respectively (de Vette, 2001).

Based on the above studies, same effect levels and low toxicity potential were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further stated that: “The studies from the two dossiers, although all rated 1, show marked difference in the results, even when the soil characteristics were similar like in the case of tests conducted with sandy loam soils. The endpoint with the lowest values is therefore selected to be taken into account, i.e., 28d EC50 = 153 mg a.i./kg dw (130 mg/kg wwt soil) and a 28d EC10 = 83 mg a.i./kg dw soil (70 mg a.i./kg ww soil), retrieved from the EQC dossier.”(ECHA biocides assessment report, 2015). Similar conclusions were drawn in the Coco TMAC biocides assessment report, 2016, where the endpoint was mainly assessed based on read across to DDAC along with the EQC owned supporting study on C12-16 ADBAC. The lowest 28d EC50 = 101.3 mg a.s. /kg dw (corrected for MW) and 28d EC10 = 59.3 mg a.s. /kg dw (corrected for MW) from the study on DDAC was selected for risk assessment.

In line with the C12 -16 biocides assessment report and gven that the read across to C12-16 ADBAC can be justified for the test substance based on a category approach, the lower 28d EC50 = 153 mg a.i./kg dw and a 28d EC10 = 83 mg a.i./kg dw soil due to inhibition of microorganisms has been considered further for hazard/risk assessment.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Hazard assessment conclusion:
PNEC aqua (freshwater)
PNEC value:
0.42 µg/L
Assessment factor:
10
Extrapolation method:
assessment factor
PNEC freshwater (intermittent releases):
0.091 µg/L
Hazard assessment conclusion:
PNEC aqua (marine water)
PNEC value:
0.042 µg/L
Assessment factor:
1 000
Extrapolation method:
assessment factor
PNEC marine water (intermittent releases):
0.009 µg/L
Hazard assessment conclusion:
PNEC STP
PNEC value:
1.1 mg/L
Assessment factor:
10
Extrapolation method:
assessment factor
Hazard assessment conclusion:
PNEC sediment (freshwater)
PNEC value:
68 mg/kg sediment dw
Extrapolation method:
equilibrium partitioning method
Hazard assessment conclusion:
PNEC sediment (marine water)
PNEC value:
6.8 mg/kg sediment dw
Extrapolation method:
equilibrium partitioning method
Hazard assessment conclusion:
no hazard identified
Hazard assessment conclusion:
PNEC soil
PNEC value:
1.66 mg/kg soil dw
Assessment factor:
50
Extrapolation method:
assessment factor
Hazard assessment conclusion:
no potential for bioaccumulation

Based on the results from the available studies with the test or read across substances, Daphnia has been identified to be the most sensitive species. The short-term 48 h EC50 value based on a read across study with C16-18 TMAC was determined to be 0.0091 mg a.i./L (nominal) and the lowest long-term 21-day NOEC value based on a read across study with C12-16 ADBAC in Daphnia was at 0.00415 mg/L (measured). Therefore, based on the available results, the test substance C16-18 and C18-unsatd. TMAC warrants a classification as ‘Aquatic Acute 1’ and ‘Aquatic Chronic 1; H410: Very toxic to aquatic life with long-lasting effects’ according to EU CLP criteria (Regulation 1272/2008/EC). M factors to be applied are 100 for acute and 1 for chronic toxicity.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion