Quaternary ammonium compounds, (hydrogenated tallow alkyl)trimethyl, chlorides

Toxicity to birds

Administrative data

Endpoint:

long-term toxicity to birds

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

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 read across 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 read across 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 read across 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 read across 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 read across substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the read across 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 read across substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the read across substance was calculated by dividing the oxygen consumption by the concentration of the read across 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 read across 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 read across 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 read across substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the read across substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the read across substance was considered further for the main study (Geerts, 2020).

 

The main study was conducted to determine the ready biodegradability of the read across 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 read across 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 read across 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 read across substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the read across 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 read across substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the read across 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 read across substance did not cause a reduction in the endogenous respiration at Day 7. The read across substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The read across substance was biodegraded by 67% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the read across substance and using a correction for the oxygen consumption by the nitrification, the read across 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 read across substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020). Based on the results of the read across study, which is a worst case read across, the test substance can also be considered to be readily biodegradable.

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 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 the CSR):

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

Cross-reference 2

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.​

Cross-reference 3

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 wt-wt 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 wet weight, 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).Based on the results of the read across study, a similar low bioaccumulation potential is expected for the test substance.

Study 3: The Bioconcentration factor (BCF) value of test substance, C16-18 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 71.94 L/Kg ww (Log BCF = 1.86). 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 wt-wt from the read across study has been considered further for hazard/risk assessment.

Cross-reference 4

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.

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Please note:A new long-term earthworm 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.

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. 

Study 3: TO ADD THE NEW LONG_TERM EARTHWORM STUDY ON C12-16 ADBAC

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 and517 mg a.i./kg soil dw has been considered further for hazard/risk assessment. 

Cross-reference 6

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 28 d 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 given 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.

Cross-reference 7

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Based on the available weight of evidence and the cationic nature, the test substance, C16-18 TMAC, is expected to have a low absorption potential followed by excretion primarily via feces. Based on QSAR predictions and data on read across substance, it is likely to undergo aliphatic hydroxylation as the first metabolic reaction. Further, based on its ionic nature, molecular weight and key physico-chemical properties it is likely to have no or very bioaccumulation potential. 

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

10

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

50

ABSORPTION:  

Oral absorption  

Based on physicochemical properties:  

According to REACH guidance document R7.C (May 2014), oral absorption is maximal for substances with molecular weight (MW) below 500. Water-soluble substances will readily dissolve into the gastrointestinal fluids; however, absorption of hydrophilic substances via passive diffusion may be limited by the rate at which the substance partitions out of the gastrointestinal fluid. Further, absorption by passive diffusion is higher at moderate log Kow values (between -1 and 4). If signs of systemic toxicity are seen after oral administration (other than those indicative of discomfort or lack of palatability of the test substance), then absorption has occurred.  

The test substance, C16-18 TMAC is an alkyl trimethyl ammonium chloride (TMAC) is a cationic surfactant, which is a UVCB with majorly C16 and C18 alkyl chain lengths and molecular weight ranging from 263.90 to 346.04 g/mol. The purified form of the substance is a creamy white solid. It has a moderate water solubility of 100 mg/L at 25 °C (based on CMC) and log Kow of 3.71 value, which was determined using estimation method based on solubility ratios.  

Based on the R7.C indicative criteria, together with the fact that the test substance is cationic with a strong adherence potential to the negatively charged surfaces of the membranes, suggests that the it is not expected to easily pass biological membranes.  

Based on experimental data on read across substances:  

A study was conducted to determine the absorption, distribution and excretion of orally administered radiolabelled read across substance, [14C] C16 TMAB (99% radiolabelled purity), in female rats. Approximately 80% of the dose of radioactivity was found in the gastro-intestinal tract 8 h after administration, only small amounts were found in the blood plasma and about 2% of the administered radioactivity was excreted in the bile during the first 12 h after treatment. The low levels of radioactivity in the serum and bile, together with the large amounts of radioactivity found in the gastro-intestinal tract, indicated poor intestinal absorption of the read across substance. Only small amounts of radioactivity were found in the liver, kidneys, spleen, heart, lungs and skeletal muscle, and the tissue radioactivity declined rapidly, only traces being found in the examined tissues 4 d after [14C] read across substance administration. Within 3 d of ingestion, 92% of the administered radioactivity had been excreted in the faeces and 1% in the urine. No radioactivity was found in the expired CO2 collected during day 1 after administration of [14C] read across substance, indicating that no complete oxidation of the cetyl group occurred. The results of thin-layer chromatography of bile and urine samples indicated that the read across substance was metabolized to some extent in the rat. Under the study conditions, the read across substance can be assumed to have very low absorption (i.e., <10%), distributed mainly in GIT and excreted in faeces (Isomaa, 1975).  

A study was conducted to determine the basic toxicokinetics of the read across substance, C12-16 ADBAC (49.9% active in water with 99.4% radiolabelled purity), according to OECD Guideline 417, in compliance with GLP. In this study, Sprague-Dawley rats were treated with single and repeated oral doses (50 or 200 mg/kg bw) as well as a single dermal dose (1.5 or 15 mg/kg bw) of the radiolabelled read across substance. Following single and/or repeated oral doses, the plasma, blood and organ radioactivity levels were essentially non-quantifiable, indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was around 8% (urine and bile fractions). This was eliminated rapidly, essentially within a 48 to 72 h period. The majority of the oral dose was excreted in the faeces. At the high oral dose level only, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines, where their levels decreased over time and were all non-quantifiable by 168 h (i.e., 7 d). Only about 4% of the oral dose was eliminated in the bile in a 24 h period, of which about 30% during the first 3 h. Under the conditions of the study and following oral administration the read across substance was found to have limited absorption (ca. 10%), low distribution (below quantification limits within 4-7 d) and majorly excreted via faeces (ca. 80%) (Appelqvist, 2006). Further, a biocides assessment report available on the read across substance by RMS Italy, concluded that the read across substance“is highly ionic and, therefore, it is expected not to be readily absorbed from the gastrointestinal tract or skin. The vast majority of the oral dose was excreted in the faeces (80%) as unabsorbed material (only about 4% of the oral dose was eliminated in the bile in a 24-hour period). The actual fraction of the oral dose absorbed was about 10%, based on the urinary mean value 3-4% (with a single peak value of 8.3%) and biliary excretion values (3.7-4.6%), as well as on the absence of residues in the carcass, as measured at 168 h. Excretion was rapid (within a 48 to 72-hour period). The radioactivity excreted in the urine was not associated with the parent compound, but with more polar metabolites which were not identified”(ECHA biocides assessment report, 2015).  

In another study conducted according to EPA OPP 85-1, Sprague-Dawley rats (10 animals per sex per group) were treated with radiolabelled read across substance, C12-16 ADBAC (30% active in water with 99.4% radiolabelled purity). The study was conducted in four phases: a single low dose (10 mg/kg); a single high dose (50 mg/kg); a 14 d repeated dietary exposure with non-radiolabelled read across substance (100 ppm) and single low dose of radiolabelled (14C) read across substance (10 mg/kg); and single intravenous dose (10 mg/kg). Following the single doses or the last dietary dose, urine and faeces were collected for 7 d. A preliminary study had indicated that insignificant 14CO2 was generated. Tissues, urine and faeces were collected and analysed for radioactivity and faeces were analysed by TLC, HPLC and MS for metabolites and parent compound. Following oral administration, radiolabelled read across substance was rapidly absorbed, although in very limited amounts, consistent with its highly ionic nature. Residual 14C in tissues was negligible after administration by gavage both after single and repeated dosing, indicating low potential for bioaccumulation. After i.v. administration a higher amount of radioactivity (30−35%) was found as residue in the tissues. About 6−8% of orally administered read across substance is excreted in the urine whereas, 87−98% was found in the faeces. Since no data on bile duct-cannulated rats are available, it was not possible to conclude if this radioactivity accounts exclusively for unabsorbed read across substance or not. However, the i.v. experiment showed that 20−30% was excreted in the urine and 44-55% in the faeces, suggesting that both the kidney and liver are capable of excreting read across substance once absorbed and that absorption is higher than the % found in the urine after oral administration. Based on the urinary mean value 3-4% (with a single peak value of 8.3%) and biliary excretion values (3.7-4.6%), as well as on the absence of residues in the carcass, as measured at 168 h, it can be expected that the read across substance absorption through the g.i. tract is about 10% (conclusion not included in the study report; as assessed by the Italian Rapporteur Member state in the biocides dossier; ECHA biocides assessment report, 2015). Less than 50% of the orally administered read across substance was found to be metabolised to side-chain oxidation products. In view of the limited absorption of the read across substance, the four major metabolites identified were expected to be at least partially formed in the gut of rats, apparently by microflora. No significant difference in metabolism between male and female rats or among the dosing regimens was observed. Repeated dosing did not alter the uptake, distribution or metabolism of read across substance. Under the conditions of the study, the read across substance was found to have limited absorption (ca. 10%; due to its ionic nature), negligible distribution (no bioaccumulation), and majorly excreted majorly via faeces (87-98%) following oral administration. However, following i.v. administration, it was found to be widely distributed (30-35%) in tissues and excreted both via faeces (40-55%) and urine (20-30%). Four major metabolites were identified, formed via oxidation of the alkyl chain (Selim, 1987). Further, the biocides assessment report concluded that“the oral absorption can be considered to be approximately 10%, based on the 5-8% of the C12-16-ADBAC administered dose eliminated via urine and tissue residues (less than 1% of the administered dose 7 days after single and repeated oral dosing). More than 90% is excreted in the faeces and the pattern did not change after repeated doses. Although it was not possible to discriminate between unabsorbed/absorbed material, based on the chemical nature of the test substance, it can be anticipated that about 90% is present in faeces as unabsorbed material. The majority of C12-16-ADBAC metabolism is expected to be carried out by intestinal flora; the metabolites, which account for less than 60% of the administered dose, include hydroxyl- and hydroxyketo- derivatives of the dodecyl, tetradecyl and hexadecyl chains. No metabolite accounted for more than 10% of the total administered dose”(ECHA biocides assessment report, 2015).  

Assessment from biocides assessment report available on read across substances:  

As indicated above the biocides assessment reports available on the read across substance C12-16 ADBAC indicated that given its ionic nature, C12-16 ADBAC was not expected to be readily absorbed from the gastrointestinal tract or skin. And based on the results from thein vivostudies with rats andin vitrostudies with human skin, an oral and dermal absorption value of 10% could be considered at non-corrosive concentrations. Another biocides assessment report by RMS Italy , on the read across substance Coco TMAC, additionally reported two supportingin vivostudies on rats from literature, apart from the studies with C12-16 ADBAC and didecyldimethylammonium chloride (DDAC), which indicated an oral uptake of C16 TMAC of about 3.3 % (1.22 excreted by urine and around 2% in bile; 92% found back in faeces on day 3); and a dermal uptake of about 3.15% (in two days: 1.76% excreted in urine, 0.28% in faeces, organs 1.11%) (ECHA biocides assessment report, 2015, 2016).  

Conclusion:Overall, based on the available weight of evidence information, the test substance can be expected to overall have low absorption potential through the oral route. Therefore, in line with the biocide assessment report and as a conservative approach a maximum oral absorption value of 10% can be considered for risk assessment.   

Dermal absorption  

Based on physicochemical properties:  

According to REACH guidance document R7.C (ECHA, 2017), dermal absorption is maximal for substances having MW below 100 together with log Kow values ranging between 2 and 3 and water solubility in the range of 100-10,000 mg/L. Substances with MW above 500 are considered to be too large to penetrate skin. Further, dermal uptake is likely to be low for substances with log P values <0 or <-1, as they are not likely to be sufficiently lipophilic to cross the stratum corneum (SC). Similarly, substances with water solubility below 1 mg/L are also likely to have low dermal uptake, as the substances must be sufficiently soluble in water to partition from the SC into the epidermis.  

The test substance is a solid paste, with an MW exceeding 100 g/mol, moderate water solubility and an estimated log Kow exceeding 3.This together with the fact that the test substance is cationic with a strong adherence potential to the negatively charged surfaces, suggests that the test substance at non-corrosive concentrations is likely to have a low penetration potential through the skin. 

At higher corrosive concentrations although there is a likelihood of exposure to the test substance due to disruption of the barrier properties of the skin, the likelihood of occurrence of these cases is expected to be minimal due to the required risk management measures and self-limiting nature of the hazard. Therefore, this scenario has not been considered further for toxicokinetic assessment.  

Based on QSAR prediction:  

The two well-known parameters often used to characterise percutaneous penetration potential of substances are the dermal permeability coefficient (Kp[1]) and maximum flux (Jmax). Kp reflects the speed with which a chemical penetrates across SC and Jmax represents the rate of penetration at steady state of an amount of permeant after application over a given area of SC. Out of the two, although Kp is more widely used in percutaneous absorption studies as a measure of solute penetration into the skin. However, it is not a practical parameter because for a given solute, the value of Kp depends on the vehicle used to deliver the solute. Hence, Jmax i.e., the flux attained at the solubility of the solute in the vehicle is considered as the more useful parameter to assess dermal penetration potential as it is vehicle independent (Robert and Walters, 2007).   

In the absence of experimental data, Jmax can be calculated by multiplying the estimated water solubility (using WATERNT v.1.02) with the Kp values from DERMWIN v2.02 application of EPI Suite v4.11. The calculated Jmax values for the different carbon chains of the UVCB substance was determined to be range between 7.33E-6 to 3.17E-03 μg/cm2/h, leading to a weighted average value of 2.53E-5 μg/cm2/h. As per Kroeset al.,2004 and Shenet al. 2014, the default dermal absorption for substances with Jmax ≤0.1 μg/cm2/h can be considered to be less than 10%. Based on this, the test substance can be predicted to have low absorption potential through the dermal route.  

Based on experimental data on read across substances:  

A study was conducted to determine the percutaneous absorption of the radiolabelled read across substance, [C14] C12 TMAB, under occlusive conditions on rat skin. The test substance was applied to the intact clipped skin of 3 rats under three scenarios: at 1% and 3% in aqueous solution followed by subsequent with and without rinsing respectively and 0.5% hair-rinse formulation of test substance. Application in a cream hair-rinse preparation under user conditions resulted in the absorption of about 0.1% of the administered radioactivity after 48 h. No measurable radioactivity was present in the blood. However, application of the test substance at 1% and 3% aqueous without subsequent rinsing solution gave a somewhat higher absorption (0.6% after 72 h and 3.15% after 48 h respectively), whereas, some radioactivity was found in the blood after application of the test substance to the skin without subsequent rinsing. Overall the percutaneous absorption of the test substance was low. Under study conditions, percutaneous absorption of the radiolabelled test substance was found to be 0.6% with rinsing and 3.15% without rinsing (Bartnik 1979).  

A study was conducted to determine thein vitrodermal absorption of the read across substance, C16 TMAC contained in a hair care formulation (14% formulation containing 25% C16 TMAC aqueous solution) using pig skin, according to OECD Guideline 428, in compliance with GLP. Preparations of dermatomed pig skin (from back and flank of castrated male pig) measuring 1000 μm in thickness with stratum corneum, epidermis and parts of the dermis were used. Six skin samples were mounted in parallel in Teflon diffusion chambers (static diffusion cell) which were continuously rinsed with receptor fluid (0.9% sodium chloride in distilled water). The test formulation containing 0.875 mg/cm2 of the read across substance was applied to the skin disks at an area dose of 25 mg/cm² (100 mg on 4 cm²) for an exposure period of 30 minutes and subsequently rinsed off with a neutral shampoo and water. Concentrations of the read across substance in receptor fluid were determined at the start of the experiment (0 h) and after 16, 24, 40, 48, 64 and 72 h by HPLC/ESI/MS detection. In addition, the read across substance was analysed in different skin layers and in the rinsing fluid in order to enable calculation of total recovery. Based on the analysis, at any of the different sampling times, small quantities of the read across substance could be detected in the horny layer (1.25-14.25 μg/cm²) and in residual skin (0.75-7.25 μg/cm², corresponding to 0.086-0.83%, with a mean of 0.27±0.28%). The total recovery was about 108%. As per the SCCS opinion, for the purposes of the risk assessment, a conservative value (limit of quantification, LOQ) for the receptor fluid may be taken as a worse case value with the assumption that the amounts in the receptor fluid were at the respective LOQ value. For the duration of 24 h, a mean value of about 3.3 μg/cm² (range 2.4-3.7 μg/cm²) can be calculated from the data in the table in Appendix III of the study. Adding the amount of the read across substance in dermis would result in 10.6 μg/cm² as a worst-case value. Under the study conditions, the worst-case dermal absorption value for risk assessment of the read across substance was assumed to be 10.6 μg/cm2 (SCCS, 2012) (i.e., equivalent to 1.2% of the applied dose).  

Following a single dermal application of the read across substance, C12-16 ADBAC in the Appelqvist (2006) study, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2 and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48-h period, suggesting that the dermal dose was highly absorbed via the skin. However, this apparent high absorption via the skin may have been due to the animal licking the test site. This was also supported with the finding that, after oral dosing, only about 4% was excreted via bile back to the intestine and 4% excreted via urine.  

If similar routes of excretion are expected for dermally absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermally applied dose was taken up orally after all. Excretion in urine (2%) following dermal exposure was similar to that following oral exposure. At 24 h post-dosing, most of the radioactivity was in the “stripped” skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestines for both dose levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels decreased over time. All data showed generally a low inter-animal variability. In addition, there was no evidence of gender differences (Appelqvist, 2006). Further, the biocides assessment report concluded that “The available data on BKC dermal absorption do not allow to quantify exactly the % of the dose which was absorbed after dermal application. However, due to the radioactivity recovered at the skin application site after removal of the stratum corneum layers (6.5-8.7% of the dose) and the ionic nature of the test item, it can be anticipated that the dermal absorption is not different from the oral one (10% at non corrosive concentration)”(ECHA biocides assessment report, 2015).   

Anin vitrostudy was conducted to determine the rate and extent of dermal absorption of the read across substance, C12-16 ADBAC (80.5% active; >99% radiolabelled purity), according to OECD Guideline 428, in compliance with GLP. The study was conducted with radiolabelled read across substance at 0.03% and 0.3% concentrations, which was topically applied over split-thickness human skin membranes mounted into flow-through diffusion cells. Receptor fluid was pumped underneath the skin at a flow rate of 1.5 mL/hour. The skin surface temperature was maintained at approximately 32 °C. A barrier integrity test using tritiated water was performed and any skin sample exhibiting a permeability coefficient (kp) greater than 2.5 x 10-3 cm/h was excluded from subsequent absorption measurements. The 14C- radiolabelled read across substance was applied at an application rate of 10 mg/cm2. Absorption was assessed by collecting receptor fluid in hourly intervals from 0-6 h post dose and then in 2-hourly intervals from 6-24 h post dose. At 24 h post dose, the exposure was terminated by washing and drying the skin. The stratum corneum was then removed from the skin by 20 successive tape strips. All samples were analysed by liquid scintillation counting. Under the conditions of the study, the mean absorbed dose and mean dermal deliveries were determined to be 0.05% (0.01 ηg equiv. /cm2) and 2.22% (0.07 ηg equivalent/cm2) of the applied dose for the low concentration test preparation, respectively, and 0.03% (0.01 ηg equivalent /cm2) and 2.16% (0.67 ηg equivalent/cm2) of the applied dose for the high concentration test preparation, respectively. The stratum corneum acted as a barrier to absorption, with the mean total unabsorbed doses (recovered in skin wash, tissue swabs, pipette tips, cell wash, stratum corneum and unexposed skin) of 96.80 and 94.68% of the applied dose for the low and high concentration test preparations, respectively. The maximum fluxes for the low and high doses were 0.12 ηg equivalent /cm2/h and 0.74 ηg equivalent /cm2/h, respectively, at 2 h (Roper, 2006). Based on literature evidence, substances with Jmax ≤ 0.1μg/cm2/h, can be expected to have low skin penetration potential and can be assigned a default skin absorption of <10% (Shenet al., 2014). Further, the dermal absorption of the read across substance was concluded in its biocides assessment report (by RMS Italy) to be 8.3%, which was obtained by summing up the radioactivity present in the receptor fluid (0.05%), at the application site (after 20 consecutive tape stripping procedures) and the one present in tape strips (n°6-20) (ECHA biocides assessment report, 2015).  

Assessment from biocides assessment report available on read across substances:  

As indicated above the biocides assessment reports available on the read across substance C12-16 ADBAC indicated that given its ionic nature, C12-16 ADBAC was not expected to be readily absorbed from the gastrointestinal tract or skin. And based on the results from thein vivostudies with rats andin vitrostudies with human skin, an oral and dermal absorption value of 10% could be considered at non-corrosive concentrations (ECHA biocides assessment report, 2015).  

Conclusion: Overall, based on all the available weight of evidence information, the test substance can be expected to have a low absorption potential absorption through the dermal route. While the studies with C16 TMAC/C12 TMAB support a lower absorption potential (<5%), as a conservative approach and in line with the biocide assessment report a maximum dermal absorption value of 10% has been considered for the risk assessment.  

Inhalation absorption  

Based on physicochemical properties:  

According to REACH guidance document R7.C (ECHA, 2017), inhalation absorption is maximal for substances with VP >25 KPa, particle size (<100 μm), low water solubility and moderate log Kow values (between -1 and 4). Very hydrophilic substances may be retained within the mucus and not available for absorption.  

The test substance, because of its relatively low vapour pressure of 2.9E-6 Pa at 25 °C, will not be available as vapours for inhalation under ambient conditions. Therefore, the substance will neither be available for inhalation as vapours nor as aerosols. Further, if at all there is any inhalation exposure, considering the moderate water solubility of the substance, it is expected to be retained in the mucus to an extent and some amount may reach the lower respiratory tract. The absorption fate of the deposited material thereafter is expected to be similar to the oral route/gastrointestinal tract.  

Conclusion: Based on all the available weight of evidence information, together with the fact that the test substance is cationic with an adherence potential to the negatively charged surfaces, suggests that the it is not expected to easily pass biological membranes. Therefore, a value of 50% can be considered for the risk assessment as a conservative approach.    

METABOLISM:  

Based on identified literature:  

As discussed in the Selim, 1987 study, less than 50% of the orally administered test substance is metabolised to side-chain oxidation products. In view of the limited absorption of the test substance, the four major metabolites identified may be at least partially formed in the gut of rats, apparently by microflora. The metabolites, which account for less than 60% of the administered dose, include hydroxyl- and hydroxyketo- derivatives of the dodecyl, tetradecyl and hexadecyl chains. No metabolite accounted for more than 10% of the total administered dose. No significant difference in metabolism between male and female rats or among the dosing regimens was observed. Repeated dosing did not alter the uptake, distribution or metabolism of the test substance (Selim, 1987).  

In addition, the even-carbon chain alkyl trimethylammonium chloride is suggested to follow degradation by a common pathway involving ω-oxidation of the alkyl chain followed by β-oxidation, to give rise to metabolites with chain lengths of C2 and C4 (SSC, 2012). Based onin vitrometabolism data identified for a cetrimonium bromide, other minor metabolic pathways, which are expected for the TMACs, involve the dealkylation of the trimethylamine and dimethylamine (Maduagwu, 1985, 1988). However, the velocity of metabolism and the formation of tertiary and secondary amines were considered to be dependent on the length and structure of the alkyl or aryl moiety of the molecule (SCCS, 2012).

Based on QSAR modelling:  

The OECD Toolbox (v.4.4.1) and FAME 3 were used to predict the first metabolic reaction, since the rat liver S9 metabolism simulator performs predictions for salts, while SMARTCyp and MetaPrint2D are not powered enough for this type of substances. The second simulator of the OECD Toolbox (in vivorat metabolism simulator) was not used as it does not consistently perform predictions for salts. As per the rat liver S9 metabolism simulator, the major constituents are primarily predicted to undergo ω or ω-1 aliphatic hydroxylation reactions. Similar results were found with FAME 2 metabolism simulation tool (which currently covers only CYP metabolism). See the below table for the reaction sites. For further details, refer to the read across justification. 

Major constituents (>=10%)		Rat liver S9 metabolism simulator / Fame 2
Cetrimonium chloride (C16)		ω or ω-1 aliphatic hydroxylation
Trimethyloctadecylammonium chloride		ω or ω-1 aliphatic hydroxylation

Overall, similar reactive sites were predicted for other TMACs and ADBACs from the category.  

Conclusion: Based on all the available weight of evidence information, the test substance is considered to be primarily metabolised by alkyl chain hydroxylation, which is carried out by the intestinal flora.   

 

DISTRIBUTION  

Based on physico-chemical properties:     

According to REACH guidance document R7.C (ECHA, 2017), the smaller the molecule, the wider the distribution. Small water-soluble molecules and ions will diffuse through aqueous channels and pores, although the rate of diffusion for very hydrophilic molecules will be limited. Further, if the molecule is lipophilic (log P >0), it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. Identification of the target organs in repeated dose studies are also indicative of the extent of distribution.    

Generally given the ionic nature of the test substance, the test substance is not likely to readily partition across the blood membranes into the different organs, leading to an overall low distribution potential. Moreover, even if the test substance distributes to a certain extent, it is not expected to bioaccumulate based on the read across experimentalBCF values of C14 TMAC or C12-16 ADBACor the predicted BCF values generated for the test substance using ionic BCF regression-based equation from BCFBAF v. 3.02 program of EPISuite^TM(see section 4.3 of the CSR).     

Based on experimental data on read across substances:    

As discussed above, in the Isomaa, 1975 study, only small amounts of radioactivity were found in the liver, kidneys, spleen, heart, lungs and skeletal muscle, and the tissue radioactivity declined rapidly; only traces being found in the examined tissues 4 d after [14C] oral administration of the read across substance in rats. In the Appelqvist, 2006 study, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 h post-dosing at 200 mg/kg bw; otherwise, the vast majority of the dose was confined to the intestines, where their levels decreased over time and were all non-quantifiable by 168 h (i.e., 7 d). In the Selim, 1987 study, residual 14C in tissues was negligible after administration by gavage both after single and repeated dosing, indicating low potential for bioaccumulation. However, following i.v. administration, it was found to be widely distributed (30-35%) in tissues (Selim, 1987). 

Conclusion:Based on all the available weight of evidence information, the test substance is expected to have a low distribution and bioaccumulation potential.  

EXCRETION: 

Based on physicochemical properties: 

Given the expected low absorption potential of the test substance due to its ionic nature and physico-chemical properties, it can be expected to be primarily excreted through faeces. 

Based on experimental data on read across substances: 

Based on the evidence from the available oral studies (Isomaa, 1975; Appelqvist, 2006; and Selim, 1987), the test substance is primarily expected in faeces (>90%) and less via urine (<10%).Further, in the Isomaa, 1975 study, no radioactivity was found in the expired CO2 collected during Day 1 after administration of [14C] C16 TMAB, indicating that no complete oxidation of the cetyl group occurred. 

Conclusion:Based on all the available weight of evidence information, the test substance is expected to be primarily excreted via faeces.        

Cross-reference 8

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

medium hazard (no threshold derived)

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

medium hazard (no threshold derived)

Most sensitive endpoint:

skin irritation/corrosion

Hazard assessment conclusion:

no hazard identified

Justification:

starting point is a NOAEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

medium hazard (no threshold derived)