Quaternary ammonium compounds, benzyl C12-C16 (even numbered)-alkyldimethyl 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

A number of reliable studies have shown that the test substance is readily biodegradable.

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Freshwater:

Study 1: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50.1% active in water) in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992).

Study 2: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80% active in hydroalcoholic solution), in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Testing at low concentrations, was required due to the toxicity of the test substance towards the inoculum at higher concentrations. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5% after 28 d (CO2 evolution). The test substance was considered to be readily biodegradable (van Dievoet, 2005). This study was also submitted as part of the biocides dossier for product type-8 and was concluded by the authority to be a key and valid study (see below discussions; ECHA assessment report, 2015).

Study 3:A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (49-52% active in water) in water according to OECD Guideline 301D (closed bottle test). Half-lives were determined using inoculum from various aquatic sources. The test substance was added to either seawater or water from the river IJseel, ditch water or mineral medium inoculated with activated sludge such that its concentration was 2 mg/dry weight/L. The oxygen decrease in the bottles as a function of time was measured using a special funnel. This funnel fitted exactly into the bottle and derverd as an overflow reservoir permitting multiple measurements in one bottle. Biochemical oxygen demands (BOD) of the test substances were corrected by subtracting the BOD of the control. The biodegradability was calculated by dividing the corrected BOD by the chemical oxygen demand (COD). The biodegradation of the test substance was 71, 69 and 60% in seawater, ditch water and river water, with half -lives of 0.3, 0.1 and 0.1 d, respectively. Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable (van Ginkel, 1996).

Study 4: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50.15% active in hydroglycolic solution) in water according to OECD Guideline 301D (closed bottle test). The method was adapted according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concerned the inoculum, the composition of the dilution water and the analyses. The inoculum was taken from an activated sludge plan, the municipal wastewater treatment plant in Duiven (NL). The sludge was preconditioned by aeration, to reduce high residual respiration rates. The density of the inoculum in the test was 3 mg s.s./L. On Days 0, 14, 28 and 42, the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled with a suspension of pre-conditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were conducted in a new series of three bottles. The test concentration was 4.3 mg/L, therefore the COD in the test suspension was 5.2 mg O2/L. After 4 weeks, the nitrite and nitrate concentrations were measured to be <0.1 and <1.5 mg/L respectively. The extent of biodegradation, calculated as the BOD related to the COD for test substance is about 65% after 2, 4 and 6 weeks. All validity criteria were fulfilled: i.e., inoculum blank indicated >1.5 mg dissolved oxygen/L after 28 days; the residual concentration of oxygen in the test bottles were >0.5 mg/L; difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or the end of the 10-d window, as appropriate, was less than 20%; and toxicity control showed >25% degradation. Therefore, under the conditions of the study, the test substance was considered readily biodegradable (Balk, 1987).

Study 5: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (50% active in water) in water according to OECD Guidelines 301D and 302A (closed bottle test / modified SCAS test), in compliance with GLP. The experiment was carried out using a combination of an inherent and a ready biodegradability test. To predict the effects of possible biodegradation products, the toxicity of effluents from semi-continuous activated sludge (SCAS) units was assessed. The test substance caused no reduction of the biodegradation of non-purgeable organic carbon (NPOC) present in primary settled sewage. Therefore, it was considered to be non-inhibitory to activated sludge. During the test period, 99% of the substance was removed from the wastewater by adsorption and/or biodegradation. In a second step, the distinction between biodegradation and adsorption was evaluated in closed bottle tests inoculated with approximately 2 mg/L of activated sludge collected on Days 0 and 28 from the SCAS unit fed with the test substance. With the Day 0 SCAS sample, the test substance was biodegraded by 52% within 28 d and by 62% within 56 d. The biodegradation in the closed bottle tests did increase due to the acclimatisation of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 28. The closed bottle test results demonstrated that the test substance was removed by biodegradation in the SCAS test. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1993). This study was primarily carried out to determine the biodegradation pathway of alkylbenzyldimethylammonium salts and not to assess the ready biodegradability; therefore, the study has been used only as a supporting study.

Study 6: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80.8% active in ethanol) in water according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. Flasks containing acclimated inoculum (at 10 mg/L) from a previous SCAS assay were dosed with 5 and 10 mg a.i./L of the test substance or 20 mg/L of the reference substance (d-glucose) and were maintained for 28 d. Biodegradability was calculated from the CO2 released over time in the test and reference flasks relative to that which was released in the blank control (a flask prepared without test or reference substance). The results indicated that 84.0 and 82.6% CO2 was produced in vessels dosed with 5 and 10 mg/L test substance, respectively, compared to 0% with the control and 80.6% with d-glucose. Final suspended organic carbon (SOC) concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose. Under the conditions of the study, the substance was considered to be readily biodegradable (Corby, 1992a).

Study 7: A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (80.8% active in ethanol) in water according to OECD Guideline 302A (Modified SCAS test), in compliance with GLP. Four chambers containing activated sludge were aerated and the suspended solid contents were adjusted to 2500 mg/L. The test substance at 1000 mg a.i./L was added to two test units for a 7-d acclimation period. This consisted of incremental additions until the final test concentration of 10 mg a.i./L was reached. Two additional units did not receive the test substance and served as control. The testing period was an additional 7 d following the acclimation period. Throughout the study, all units were fed synthetic sewage. Effluents withdrawn from each unit were analysed for SOC. Under the conditions of the study, the average percent SOC removal was >100% indicating that the test substance was inherently biodegradable (Corby, 1992b).

A literature review was conducted to determine the biodegradability of the test substance, C12 -16 ADBAC (purity not specified). Van Ginkel et al 2004 and Patrauchan et al 2003 publication were reviewed. Based on the literature data, the test substance is considered readily biodegradable (Van Ginkel, 2004 and Patrauchan, 2003).

The Biocides assessment report on C12-16 ADBAC, published by the Italian authorities in June 2015, reported the above key studies and stated that“The reliability factor of US ISC study (van Dievoet, 2005)is 1. Therefore, the study by US ISC should be considered for the environmental risk assessment at product authorization stage. In conclusion, ADBAC/BKC is ready biodegradable being the 10-day window criterion met (OECD 301B). On the other hand, the EQC study (van Ginkel and Stroo, 1992) has a reliability factor of 2 because it cannot distinguish between the degradation of ADBAC/BKC and Propan-2-ol (solvent). If we follow the argument that Propan-2-ol is readily biodegradable and might contribute more to the oxygen consumption. This results in an overestimation of ADBAC/BKC, and the 14-day window criteria was not met (OECD 301D). Alkyl (C12-16) dimethylbenzyl ammonium chloride is readily biodegradable.”  

Sea water:

A study was conducted to determine the biodegradation of the test substance, C12-16 ADBAC (49-52% active in water) in seawater and sediment according to OECD Guideline 306 (biodegradation in seawater). Three bottles containing only seawater and 3 bottles containing seawater and the test substance were used. The test substance was added at a concentration of 2 mg/L. The biodegradability was determined by following the course of the oxygen decrease in the bottles using a special funnel. The funnel fitted exactly in the bottle and served as an overflow reservoir permitting multiple measurements in one bottle. The oxygen concentration was measured on Days 0, 7, 14, 21. 28. 42, 56 and 84. The test substance was toxic to microorganisms and was therefore studied in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance should be released slowly from the silica gel (0.5 g/bottle). Although no additional oxygen consumption was expected, controls with silica gel were carried out as well. Under the study conditions, the test substance was biodegraded by 38 and 31% on Day 28 in the absence and the presence of silica gel, respectively. Since the test substance was biodegraded at 61% on Day 84 in the prolonged closed bottle test with silica gel, it is expected to be biodegraded in seawater (van Ginkel, 1994).

 

Therefore, based on the available information and in line with the biocides assessment report, the test substance is considered to be readily biodegradable.  

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Based on the most recent and radiolabelled aerobic biodegradation study in soil, the transformation of the C12 and C14 carbon chains of the test substance was considered to be rapid with DT50 values ranging from 2.2-28.7 days with the SFO model and 1.6 – 23.3 days with the FOMC model at 20°C.​ Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error was used further for risk assessment.

Therefore, in line with the biocides dossier, the DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) also has been considered further for hazard/risk assessment.

Half-life in soil:

17.1 d

at the temperature of:

12 °C

Study 1:  

A study was conducted to determine the aerobic transformation/dissipation in the soil of the test substance, C12 -16 ADBAC (radiochemical purity: 98.5%), according to the OECD Guideline 307, in compliance with GLP. Four different standard soils (LUFA 2.2, 2.3, 2.4 and 5M, field fresh sampled), varying in their organic carbon content, pH, clay content, cation exchange capacity and microbial biomass, were treated with [ring-U-14C] Benzalkonium chloride. Soil samples were incubated in the dark under aerobic conditions for up to 128 days under controlled laboratory conditions. After appropriate time intervals, soil samples were extracted, and the extracts were analysed for test substance and transformation products to calculate DT50 and DT90 values. The mineralization was determined by trapping and analysis of the evolved 14CO2. Non-extractable residues (NER) were determined after combustion of the extracted soil samples. The total radioactivity of the soil extracts, the extracted soil (NER) and evolved 14CO2 was determined by LSC. Test substance and transformation products in the soil extracts were analysed by LC-FSA (radio-HPLC). Evaluation of the transformation pathway was done by LC-HRMS. 

Transformation of the C12 chain of the test substance [ring-U-14C]Benzalkonium chloride was rapid in all four soils. The transformation of the C14 chain started after a short adaptation phase but was thereafter rapid as well. Within 7 - 21 days the concentration of the C12 chain decreased from initially 67.2 – 69.6% of applied radioactivity (AR) to < 20 % of AR. The concentration of the C14 chain decreased from initially 23.8 – 24.6 % of AR to < 10 % of AR within 10 – 36 days. Formation of NER started directly after application of the test substance. Further formation of NER increased in parallel to the start of increased mineralisation, indicating that a major amount of NER is comprised by radioactivity incorporated in microbial biomass. At the test end, the biomass concentration was in the range of 1.46 – 2.62 % of soil organic carbon content in all four soils, indicating that viable microbial biomass was present throughout the incubation time. The mass balance was in the range 99.9 – 103.0 % at test start and 90.4 – 94.0 % at test end.

The predominant initial degradation step was the oxidative removal of the alkyl chain. Dimethylbenzylamine was determined as the major metabolite, the highest concentrations of dimethylbenzylamine were determined until Day 22, thereafter the concentrations deceased continuously until test end. Methylbenzylamine was transient and only present in traces. Benzylamine, a suspected metabolite, was not detected. Further metabolites containing partly degraded alkyl chains were all transient and were not detected or only <0.2 % of AR (soil 2.3) at the test end.

With regard to the kinetics, the transformation showed a slight bi-phasic pattern, therefore the ‘Single First Order Model’ (SFO) and the ‘First-Order Multi-Compartment Model’ (FOMC) were compared. Based on the visual fit and x2 error, the transformation of [ring-U-14C]Benzalkonium chloride met the requirements for both models well for all four soils. The calculated DT50 values with the Single-First-Order Model (SFO) for the dissipation of [ring-U-14C]Benzalkonium chloride were 2.2 – 8.7 days (C12 chain) and 6.1 – 28.7 days (C14 chain), the DT90 values were 7.2 – 28.8 (C12 chain) days and 20.2 – 95.4 days (C14 chain). The calculated DT50 values with the FOMC model for the dissipation of [ring-U-14C]Benzalkonium chloride were 1.6 – 7.2 days (C12 chain) and 5.5 – 23.3 days (C14 chain), the DT90 values were 15.0 – 48.8 days (C12 chain) and 35.8 – 164.3 days (C14 chain).

The test substance is predominantly C12-ADBAC and C14-ADBAC, with low to negligible amounts of C16-ADBAC. The chain length distribution is defined as follows:C12 (35-80%), C14 (20-55%), C16 (0-15%). C16-ADBAC was not included in this study because it is present in very low amounts; there are technical difficulties with having sufficient radioactivity for substances present in small amounts relative to other constituents. C16-ADBAC would be expected to degrade by the same route but at a slower rate than its C12 and C14 counterparts, as degradation rate tends to decrease with increasing chain lengths. Under the study conditions, transformations of both C12 and C14 carbon chains of the test substance were determined to be rapid in all four soils and the DT50 values were determined to be 2.2 – 8.7 days [C12 chain] and 6.1 – 28.7 days [C14 chain] with the SFO model and 1.6 – 7.2 days [C12 chain] and 5.5 – 23.3 days [C14 chain] with the FOMC modelat 20°C (Fiebig, 2019).

Further, in the biocides dossier, to account for the potential contribution of C16 ADBAC to the overall DT50 of ADBAC, a geometric mean of SFO and FOMC DT50s for C12 and C14 ADBAC in the four soils (as recommended in BPR Vol IV Part B and C) was calculated and converted to 12° using the following equation (DT50 (12°) = DT50 (20°) * e^{(0.08*(20-12))}. This was followed by linear extrapolation of the geometric mean DT50s for C12 and C14 ADBAC, to estimate the DT50 for C16 ADBAC. See tables below:

SFO DT50s
	Soil	2.2	2.3	2.4	5M	Geo. Mean	Adj. to 12° C
	C12 ADBAC	2.2	3.3	6.2	8.7	4.4	8.4
	C14 ADBAC	6.1	8.9	12.9	28.7	11.9	22.6
	C16 ADBAC	--	--	--	--	--	36.7

 

FOMC DT50s
	Soil	2.2	2.3	2.4	5M	Geo. Mean	Adj. to 12° C
	C12 ADBAC	1.6	3.2	5.8	7.2	3.8	7.3
	C14 ADBAC	5.5	8.3	12.1	23.3	10.7	20.2
	C16 ADBAC	--	--	--	--	--	33.1

 

A weighted estimate of the DT50 of ADBAC (C12-C16) at 12°C was calculated by assuming the highest allowable concentrations of C14- and C16- ADBAC and the balance of C12-ADBAC (i.e., 12% C16, 52% C14 and 36% C12), which resulted in the following estimated DT50s:

SFO DT50 = 19.2d at 12°C; FOMC DT50 = 17.1d at 12°C

However, due to the relatively low levels of C16-ADBAC, the overall estimated DT50s were considered rather insensitive to the assumed DT50 for C16-ADBAC. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error was used further for risk assessment.

Study 2: A study was conducted to determine the aerobic biodegradation of the test 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 test 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 test 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 test 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 test substance after 70 days. This percentage of the theoretical carbon dioxide production presumes complete mineralization. The DT50 was estimated to be 40 days (van Ginkel, 1994).  

Based on the most recent and radiolabelled aerobic biodegradation study in soil, the transformation of the C12 and C14 carbon chains of the test substance was considered to be rapid with DT50 values ranging from 2.2-28.7 days with the SFO model and 1.6 – 23.3 days with the FOMC model at 20°C.​ Further, in the biocides dossier, a weighted estimate of the DT50 value at 12°C was extrapolated for C12-16 ADBAC by assuming the highest allowable concentrations for the major chains. These calculations resulted in the estimated FOMC DT50 of 17.1 days at 12°C and SOF DT50 of 19.2 days at 12°C. The DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) showing better visual fit and lower error was used further for risk assessment.

Therefore, in line with the biocides dossier, the DT50 of 17.1 days at 12°C based on the biphasic model (FOMC) also has been considered further for hazard/risk assessment.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Given the ionic nature of the test substance and the available experimental and predicted BCF values indicates a low bioaccumulation potential. The experimental BCF value of 79 L/kg ww of the test substance 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 test 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 test 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 test 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 study conditions, the whole body BCF of the test substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989).

Study 2: The Bioconcentration factor (BCF) value of test substance, C12 -16 ADBAC 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 value using this method is generally considered to be more realistic or accurate. However, ionic, pigments and dyes, perfluorinated substances are currently excluded from the applicability domain of this model. In the case of the test substance, considering that it is a UVCB consisting of a mix of ionic (e.g., the quaternary ammonium salts) and non-ionic constituents (e.g., amines), the BCF values were predicted using the regression-based method for the ionic constituents and the Arnot-Gobas BAF-BCF method for the non-ionic constituents, using SMILES codes as the input parameter. The BCF values for the constituents ranged from 1.55 to 70.8 L/kg ww (log BCF: 0.19 to 1.85), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or the 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 69.62 L/Kg ww (Log BCF = 1.84). 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. 

Study 3: A study was conducted to determine the biomagnification (BMF) potential of the read across substance, C18 TMAC (purity 95%), following the principles of OECD TG 305. For the main study rainbow trout (Oncorhynchus mykiss) with an average weight of 5.42 g were fed test diets enriched with read across substance (23.6 mg/kg read across the substance in feed. The resulting treatment and one control group (each 40 animals) were tested simultaneously. The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. The concentrations of the read across substance in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. Chemical analysis of the read across substance was performed by liquid chromatography with coupled mass spectrometry (LC-MS/MS). In the main study five animals of each group were sampled randomized on Day 7 and Day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Biomagnification factor (BMF) and distribution factor were calculated based on the tissue concentrations measured at the end of the uptake phase. No mortality or abnormal behaviour of the test animals was observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish was euthanized at Day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95. Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with the read across substance. No statistically significant difference was detected with regard to the growth of the treated groups. Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated as 0.02709 g/g, which showed that read across substance did not biomagnify after dietary exposure. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. The kinetic BMF (BMFk) and growth-corrected biomagnification factor (BMFkg) were calculated for the read across substance to be 0.0404 and 0.0463, respectively. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside from the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis. Under the study conditions, the read across substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively, suggesting low biomagnification potential (Schlechtriem, 2021). Based on the results of the read across study, a similar low biomagnification potential is expected for the test substance. 

This is further supported by the absence of 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 TM AC's assessment ((ECHA biocides assessment report, 2015, 2016).

Overall, given the ionic nature of the test substance and the available experimental and predicted BCF values indicates a low bioaccumulation potential. The experimental BCF value of 79 L/kg ww of the test substance 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, the 16-d EC50 value of 277 mg a.i./kg soil dw obtained for Brassica alba (mustard) due to effects on growth has been considered further for hazard/risk assessment. Additionally, in accordance with the ECHA R.7c guidance (2017), the 16-d NOEC value of 856.2 mg a.i./kg soil dw obtained forTrifolium pratense(red clover) due to effects on growth can be considered as the long-term equivalent value.

Short-term EC50 or LC50 for terrestrial plants:

277 mg/kg soil dw

Long-term EC10, LC10 or NOEC for terrestrial plants:

856.2 mg/kg soil dw

Study 1:A study was conducted to determine the long-term toxicity of the test 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) andTriticum 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 test 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 test 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 test 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. 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 Under the conditions of the study, based on effect on growth, mustard was identified to be the most sensitive species with lower EC50 value of 277 mg a.i./kg soil dw (Gray, 2004).l 

Study 2:A study was conducted to determine the toxicity of the test 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) andTriticum aestivum(wheat) were used. Using 0.5 L capacity plastic pots, the test 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 test 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 LC50 values in natural soil based on effect on emergence were 3881, >4990 and >4990 mg a.i./kg soil dw for mustard, red clover and wheat respectively; while those in sand were 130, 197, 234 mg a.i./kg soil dw. The corresponding NOEC values were 2772.2, 856.2, >4990 mg a.i./kg soil dw in natural soil and 55.8, 93.4 and 93.4 mg a.i./kg soil dw in sand. The EC50 values in natural soil, based on the effect on growth were 342, 309, 684 mg a.i./kg soil dw (based on changes in wet weight) and 537, 634 and 1960 mg a.i./kg soil dw (based on changes in dry weight) for mustard, red clover and wheat respectively, respectively; while those in sand were 31, 19, 105 mg a.i./kg soil dw (based on changes in wet weight) and 73, 74 and 141 mg a.i./kg soil dw (based on changes in dry weight) 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, based on effect on emergence and wet weight changes (growth) red clover was identified to be the most sensitive species with lower NOEC and EC50 value of 856.2 and 309 mg a.i./kg soil dw respectively (Servajean, 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 wasBrassica albawith an EC50 = 277 mg/kg soil dw (US ISC), which is the endpoint to be taken into account at product authorization stage (ECHA biocides assessment report, 2015).   

In line with the C12 -16 ADBAC biocides assessment report, the 16-d EC50 value of 277 mg a.i./kg soil dw obtained forBrassica alba(mustard) due to effects on growth has been considered further for hazard/risk assessment. Additionally, in accordance with the ECHA R.7c guidance (2017), the 16-d NOEC value of 856.2 mg a.i./kg soil dw obtained forTrifolium pratense(red clover) due to effects on growth can be considered as the long-term equivalent value. 

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Based on the results of short-term toxicity studies, the 14 d LC50 has been selected to express the acute toxicity of the test substance. Further, based on the chronic toxicity study the 28-d NOEC of 125 mg/kg bw/day has been considered further for hazard/risk assessment.  

Short-term EC50 or LC50 for soil macroorganisms:

7 070 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

125 mg/kg soil dw

Short-term toxicity studies: 

Study 1. A study was conducted to determine the toxicity to soil macroorganisms of the test 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 test 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 test 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 test 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 soil dw (or > 517 mg a.i./kg soil 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.”  

 Long-term toxicity study: 

A study was conducted to determine the effects of test substance (50% active in water) on mortality, biomass and the reproductive potential of the earthworm speciesEisenia fetida(Annelida, Lumbricidae), according to the OECD TG 222, in compliance with GLP. The study was conducted under static conditions over 8 weeks with the test substance concentrations 125, 250, 500, 1000, 2000 mg//kg solid dry weight (SDW) corresponding to 62.5, 125, 250, 500, 1000 mg a.i./kg SDW. Each application rate was mixed into artificial soil containing 5% peat. A control including untreated artificial soil was tested under the same conditions as the test substance treatments. A total of 80 test organisms were divided equally into 8 control replicates adnd another total of 40 test organisms were divided equally into 4 replicates for each test substance treatment (i.e., 10 earthworms per replicate). They had an individual body weight between 0.36 and 0.55 g at the experimental starting. Each concentration level and control were analysed via LC-MS/MS analysis on Day 0, Day 28 and Day 55 using pooled samples of all replicates. The measured concentrations of the pooled samples of replicates were within the range of 83 to 101 % of the nominal values on Day 0, demonstrating the right preparation of the tested concentrations. After 28 days of exposure in soil, no test substance-related earthworm mortalities (<10%), pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all test substance concentrations. There were no statistically significant differences in earthworm body weights in all test substance concentrations compared to the control. After an additional 4 weeks, the reproduction rate (average number of juveniles produced) was 83 juveniles in the control and ranged from 18 to 74 juveniles in the test substance treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 125 and 250 mg test substance/kg SDW compared to the control. However, at the test substance concentrations 500 to 2000 mg test substance/kg SDW the earthworm reproduction was statistically significantly reduced. All validity criteria recommended by the test guidelines were fulfilled. Under the study conditions, the LOEC (mortality, biomass), NOEC (mortality, biomass), LOEC (reproduction), NOEC (reproduction) and EC50 (reproduction) values for test substance were reported to be >2000, ≥2000, 500, 250 and 589 mg test substance/kg SDW, respectively (equivalent to >1000, ≥1000, 250, 125 and 295 mg a.i./kg SDW, respectively). 

Therefore, based on the results of short-term toxicity studies and in line with the biocides assessment report, the 14 d LC50 of 7070 has been selected to express the acute toxicity of the test substance. Further, based on the chronic toxicity study the 28-d NOEC of 125 mg/kg bw/day has been considered further for hazard/risk assessment.    

Cross-reference 6

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Based on the study results, the 3 h EC50 and NOEC values of the test substance for toxicity to micro-organisms were determined to be 7.75 and 1.6 mg a.i./L (measured) respectively; these values have been considered further for hazard/risk assessment.

EC50 for microorganisms:

7.75 mg/L

EC10 or NOEC for microorganisms:

1.6 mg/L

Study 1. A study was conducted to determine the toxicity to microorganisms of the test substance, C12-16 ADBAC (49.9% active in water) according to OECD Guideline 209, in compliance with GLP. The respiration inhibition test was performed on activated sludge fed with a standard amount of synthetic sewage. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The validity of the test was shown by two criteria. First, the control respiration rates were within 15% of each other and secondly, the EC50 of the reference compound 3, 5-dichlorophenol (5.2 mg/L) was within the prescribed range of 5 to 30 mg/L. No analytical dose determination was performed. Under the study conditions, the 3 h EC50 of the test substance for activated sludge was 11 mg a.i./L (with 95% confidence limits of 3 and 27 mg a.i./L). The 3 h EC10, EC20 and EC80 were 4, 5 and 24 mg a.i./L, respectively (Geerts, 2004).

Study 2. A study was conducted to determine the toxicity to microorganisms of the test substance, C12 -16 ADBAC (49 -51% active) according to OECD Guideline 209, in compliance with GLP. A mixture of activated sludge, synthetic sewage feed and a range of concentrations of the test or reference substance were prepared in 1 L glass beakers. The mixtures were aerated and, after an incubation period of 3 h at a temperature between 21.9 and 23°C, the decrease in the oxygen concentration in the mixtures was recorded during a period of 10 min. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. The substance was tested at nominal concentrations of 0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (equivalent to ca. 0, 0.5, 1.6, 5.0, 16, 50, 160 and 500 mg a.i./L). No analytical dose determination was performed. A control test with reference substance 3,5-dichlorophenol yielded an EC50 value of 5 mg/L, which is within the range of prescribed by guideline. The validity criteria of the guideline were fulfilled. Under the study conditions, the 3 h EC20, EC50 and EC80 were determined to be 6.8, 15.5 and 35.5 mg/L respectively (equivalent to 3.4, 7.75 and 17.8 mg a.i./L, respectively). The 3 h NOEC was established at 3.2 mg/L (equivalent to 1.6 mg a.i./L) (Mayer, 2001).

Based on the above studies, the same effect levels were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. Both the studies were judged reliable, but the results of the test conducted for a longer period (3h) was considered to be more appropriate as it also provides the lowest endpoints, hence the US ISC data (Mayer, 2001) was selected as the key value (ECHA biocides assessment report, 2015).Therefore, in line with the biocides assessment report on C12-16 ADBAC by RMS Italy (ECHA biocides assessment report, 2015), 3 h EC50 and NOEC values of 7.75 and 1.6 mg a.i./L (measured) respectively has been considered further for hazard/risk assessment.

Cross-reference 7

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Toxicokinetics data from studies conducted under in vitro and in vivo conditions suggests that the test substance, C12-16 ADBAC, has a low bioaccumulation potential and only a small fraction of the test substance is likely to be absorbed and distributed over the body. Therefore, a 10% absorption factor was considered for the chemical safety assessment for both oral as well as dermal routes as a worst-case approach even though with the most relevant and valid studies, a 1% dermal absorption would be a high estimate. An absorption of 100% was considered for the inhalation route.

The available data for the test substance on dermal absorption does not allow the quantification of the dose which was absorbed after dermal application. However, based on 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 substance, it can be anticipated that the dermal absorption is not different from the oral one (10%). The primary effect involves disruption of the cytoplasmic membrane causing cell damage or lyses of the cell content. Due to adherence to negatively charged surfaces of the apolar alkyl chain, ADBAC substances will not easily pass biological membranes. Dermal uptake is therefore very limited at low, non-irritating concentrations.

Bioaccumulation potential:

low 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. 

C12-16 ADBACis an alkyl dimethyl benzyl ammonium chloride (ADBAC) type of cationic surfactant. It is a UVCB with majorly C12 to C16 alkyl chains with molecular weight ranging from 283.9 to 424.02 g/mol. The purified form of the substance is a crystalline, hygroscopic, sticky white solid. It has a moderate water solubility of 500-1000 mg/L at 20°C (based on CMC) and a low log Kow of 2.75 value, which was determined 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 it is not expected to easily pass biological membranes.

Based on experimental data on read across substances:  

A study was conducted to determine the basic toxicokinetics of the test 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 test 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, most 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. Following a single dermal application, 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 is also supported by 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 the intestine with only 2% excreted via urine. This indicates that about 50% of the dermally applied dose was taken up orally after all. According to the same oral kinetics, this leads to the 2% excretion in urine as indeed was observed. 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. Under the study conditions and following oral administration the test 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%). The results following dermal application are considered to be invalid, as the experiment suffered from design flaws, allowing for oral uptake from the skin after the 6 h exposure period (Appelqvist, 2006).

Further, a biocides assessment report available on the test 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 test substance, C12-16 ADBAC (30% active in water with 99.4% radiolabelled purity).Sprague-Dawley rats (10 animals per sex per group) were treated with a radiolabelled test substance. 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 test substance (100 ppm) and a single low dose of radiolabelled (14C) test 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 test 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 test 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 the unabsorbed test 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 test 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 test substance absorption through the g.i. tract is about 10% (a 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 test substance was found to be metabolised to side-chain oxidation products. Given the limited absorption of the test 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 test substance. Under the conditions of the study, the test 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 the test substance:  

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 the available weight of evidence information, the test substance at non-corrosive concentrations 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 Kow 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 crystalline, hygroscopic, sticky white solid with an MW exceeding 100 g/mol, moderate water solubility and an estimated log Kow above 2.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 predictions:  

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 5.00E-07 to 8.50E-05 μg/cm2/h, leading to a weighted average value of 5.07E-06 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:  

Following a single dermal application of the test 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).  

An in vitro study was conducted to determine the rate and extent of dermal absorption of the test 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 test 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 were excluded from subsequent absorption measurements. The 14C- radiolabelled test 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, Kroeset al.,2004). Further, the dermal absorption of the test 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). 

 

Another in vitro study was conducted to determine the dermal absorption of the test substance, C12 -16 ADBAC (25.5% active in water; radiochemical purity: >98%) according to a method comparable to OECD Guideline 427, in compliance with GLP. The dermal absorption and excretion study was conducted in rats following application of 0.4 mL of a 0.77% w/w solution of the test substance over approximately 20 cm2 of shaved skin, under a gauze patch for 72 h. After a single topical application of radio-labelled test substance, the total amount of radioactive substance was 16% in males (urine 0.8%, faeces about 9.9% and carcass about 5.3%) and 14% in females (urine about 0.7%, faeces about 6.1% and carcass about 7.0%). This was equivalent to a total mass of 24 µg equivalents (males) and 21 µg equivalents (females) absorbed per cm2(after a dose of approximately 3 mg). Most of the radio-labelled test substance (62.6%, males; 63.2%, females) was found in both the treated (48.0%, males; 45.1%, females) and the untreated (14.6%, males; 18.1%, females) skin after 72 h. The radioactive substance in the untreated skin may have been due to surface migration of the applied material from the perimeter of the treated area. The overall recoveries of radioactivity were acceptable for the experimental objectives of quantifying the absorption of radio-labelled test substance after a single dermal application. Under the study conditions, the findings indicate that the dermal absorption of the test substance is limited and most of the absorbed test substance is excreted in the faeces (Hallifax, 1991).

Additionally, a publication of Blank (1964) was identified which reported an in vitro study evaluating the dermal penetration of the test substance, C12-16 ADBAC (purity not specified) in normal excised human skin. From un-buffered aqueous solutions of the test substance ranging in concentration from 0.005 (1.7 ppm) to 0.1 M (34 ppm), no measurable amount penetrated the dermis of excised human skin within periods of 1 to 3 d at temperatures between 23 and 35°C. Lowering the pH of the contact solution up to pH 1.3 had no influence. However, at pH 10.5 to 12, the test substance could be recovered from the skin. At these levels, electrical conductivity indicated damage to the cutaneous barrier. Similarly, pre-treatment of skin at that pH caused damage to the skin and resulted in penetration of the test substance upon subsequent contact to test solutions. Also, damaging the skin by repeated stripping of the stratum corneum with pressure-sensitive tape resulted in penetration into the skin when stripped more than 10 times (Blank, 1964).

A corneal penetration was identified from literature sources, where single or multiple drops of radiolabelled test substance, C12 -16 ADBAC (0.03% radiolabelled purity) was instilled on the cornea of rabbits to determine the corneal penetration. The test substance was found in the palpebral and bulbar conjunctiva, corneal epithelium, stroma and endothelium. Single-drop administration resulted in high tissue levels in the anterior ocular tissues that were retained for up to 120 h. Multiple-drop administration led to accumulation in the epithelium to a greater degree than any other tissue. However, at no time did the test substance appear in the aqueous humour or any other tissue besides cornea and exposed conjunctivae (Green, 1986).

Assessment from biocides assessment report available on the test substance: 

As indicated above the biocides assessment reports available on the test 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 at non-corrosive concentrations can be expected to have a low absorption potential absorption through the dermal route. As a conservative approach and in line with the biocide assessment report a maximum dermal absorption value of 10% can be considered for 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 crystalline, hygroscopic, sticky white solid physical state and relatively low vapour pressure of < 5.8E-3 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. In the case of spraying applications, coarse droplets would be formed which typically settle on the ground and result in a very lower inhalable or respirable fraction. Of the inhalable fraction, due to the droplet size and the moderate water solubility, almost all droplets are likely to be retained in the mucus and will not be available to reach the deeper lungs. The deposited droplets in the upper respiratory tract are expected to be absorbed in a relatively slower rate compared to the deeper lungs due to differences in vascularity. Some amounts of these deposited droplets are also expected to be transported to the pharynx and swallowed via the ciliary mucosal escalator. Therefore, the systemic uptake of the test substance via the respiratory route can be considered to be similar to the oral route.

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, the test substance at non-corrosive concentrations can be expected to have a low to moderate absorption potential through the inhalation route, depending on the droplet size. Therefore, a value of 50% can be considered for the risk assessment.           

.  

METABOLISM:  

Based on experimental data on test substances:  

As discussed in the Selim, 1987 study, less than 50% of the orally administered C12-16 ADBAC is metabolised to side-chain oxidation products. Given 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 hydroxy keto- 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). 

Based on QSAR modelling: 

The OECD Toolbox (v.4.4.1) and FAME 3were 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 substance. 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 3 metabolism simulation tool (which currently covers only CYP metabolism). See the table in the CSR for the reaction sites. For further details, refer to the read-across justification.

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 is 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 experimentalBCF values of C12-16 ADBAC(see section 4.3 of the CSR). 

Based on experimental data on read across substances: 

As discussed above, 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).  

 

A couple of studies were also identified from literature sources (Bogs, 1971; Cutler, 1970):

The Bogs (1971) article reported a study evaluating the distribution of the test substance, C12-16 ADBAC (purity not specified) inside the body of rabbits, cats and dogs. A single high dose (approximately 1 mL of 15% solution of the test substance in water/kg bw of animals, which is equal to about 10 times the lethal dose) was administered by oral, rectal and intramuscular route. Within a few minutes, the animals died, and the test substance concentrations were measured locally at the sites of dosing, in blood, liver and kidneys. It was concluded that only a small fraction is absorbed and distributed in the body. Less than 1.5% of the applied dose was found in the organ investigated (sites of dosing, blood, liver and kidneys), of which the major part was found in the liver (Bogs, 1971).

The Cutler (1970) article reported studies conducted in rat and Beagle dogs, which compared the application of test substance, C12 -16 ADBAC (purity not specified) in both milk and water as vehicle. Rats received 50 and 100 mg/kg bw/day for 12 weeks, and dogs 12.5, 25 and 50 mg/kg bw/day for 52 weeks. Depression in weight gain was observed in rat receiving 100 mg/kg bw/day in the water, but not in milk. Mortality occurred in dogs at 25 and 50 mg/kg bw/day in the water, but not in milk. The 12.5 mg/kg bw/day dose level was well tolerated (Cutler, 1970).

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 which is 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 (Appelqvist, 2006; and Selim, 1987), the test substance is primarily expected in faeces (>90%) and less via urine (<10%). 

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

[1] Log Kp = -2.80 + 0.66 log kow – 0.0056 MW 

Cross-reference 8

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Based on the results of the read across studies with C12-16 ADBAC, no toxicologically significant systemic toxicity is expected for the test substance. In line with the biocides assessment report, it was concluded that all effects could be attributed to local gastrointestinal irritation/corrosion and consequently reduced food intake without observing any primary systemic effect. Therefore, selection of critical NOAEL and the derivation of a systemic NOAEL or DNEL was deemed inappropriate  

Toxic effect type:

concentration-driven

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

45 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

dog

Quality of whole database:

The information requirement for this tonnage band is sufficiently met with the available data.

System:

other: no true systemic effects

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Oral:

Study 1:

Dose range-finding: A 14-day range-finding study was conducted to determine the dose levels for a 90-day repeated dose oral toxicity of the read across substance, C12-16 ADBAC (48.9% active) in Sprague-Dawley rats, according to OECD Guideline 407, in compliance with GLP. In this study, the read across substance was administered to six rats per sex per group at dietary doses of 0, 1250, 2500 and 5000 ppm i.e., equivalent to 0, 650, 1250 and 2500 ppm a.i. or 0, 112, 229 or 436 mg/kg bw/d for males and 0, 116, 229 or 427 mg/kg bw/d for females. Besides lower food intake with related lower increase of body weight gain in all groups, which was due to the palatability of the read across substance, no toxic effects were observed. Under the study conditions, the NOAEL for systemic toxicity was > 2500 ppm (i.e., equivalent to 436 and 427 mg/kg bw/day or 218 and 214 mg a.i./kg bw/d for males and females, respectively) (Chevalier, 2002).

Main study: A 90-day study was conducted to determine the repeated dose oral toxicity of the read across substance, C12-16 ADBAC (48.9% active in water) in Sprague-Dawley rats according to OECD Guideline 409, in compliance with GLP. In this study, the read across substance was administered to ten rats per sex per group, at dietary doses of 0, 400, 1000 and 2500 ppm (equivalent to 0, 28, 68 and 166 mg a.i./kg bw/day for the males and 0, 30, 74 and 188 mg a.i./kg bw/day for the females, based on food consumption and body weight information). No signs indicative of toxicity was observed in any group. At 2500 ppm, the only effect was a decrease in body weight gain (statistically significant) correlating with lower food consumption due to the low palatability of the read across substance. Further, some statistically significant deviation from control in haematological and clinical-chemistry values were observed. However, in the absence of a dose-response relation, these effects were considered to be of no clinical significance. Under the conditions of the study, the rat NOEL for systemic effects was established at 1000 ppm (i.e., equivalent to 68 and 74 mg a.i./kg bw/day for males and females, respectively) (Chevalier, 2002).

 

Study 2: A 90 d study was conducted to determine the repeated dose oral toxicity of the read across substance, C12-16 ADBAC (79.7 -80.5% active) in Sprague Dawley rats, according to OECD Guideline 408 and US EPA OPP 82 -1, in compliance with GLP. In this study, the rats were administered daily dietary levels of 0, 100, 500, 1000, 4000 and 8000 ppm read across substance, equivalent to 0, 6, 31 and 62 mg/kg bw/day (i.e. 0, 4.8, 25 and 50 mg a.i./kg bw/day) (males) and 0, 8, 38 and 77 mg/kg bw/day (i.e. 0, 6.4, 30 and 62 mg a.i./kg bw/day) (females) for 95 and 96 days, respectively. Daily intakes at 4000 and 8000 ppm could not be calculated due to high mortality. The animals were observed for mortality, clinical signs, body weight, food consumption, hematology and clinical chemistry at termination. Gross and histopathological examinations were also performed. Other than a slight trend in reduced body weight and food consumption in males at 1000 ppm, there were no treatment-related findings at 1000 ppm or less. The highest dose of the read across substance led to 100 and 80% mortality for 8000 and 4000 ppm group respectively indicating 1000 ppm to be the maximal tolerated dose. The animals that survived at 4000 ppm were cachectic and debilitated. The probable cause of death was assumed to be shock secondary to fluid and/or ionic shifts in the gastro-intestinal tract, which was attributed to irritation and corrosivity properties of the substance. The females showed less aberrations in all measurements than the males. Based on the decreased food consumption and body weights at 1000 ppm, the NOEL for the read across substance was established at 500 ppm in the diet, i.e., equivalent to 31 mg/kg bw/day (i.e., 25 mg a.i./kg bw/day) for males and 38 mg/kg bw/day (i.e., 30 mg a.i./kg bw/day) for females (Van Miller, 1988).

Study 3:

Dose range-finding: A 14-day range finding study was conducted to determine the dose levels for a 28-day repeated dose oral toxicity of the read across substance, C12 -16 ADBAC (purity 49.9%) in Beagle dogs, according to OECD Guideline 407. In Phase I, four incremental doses of 500, 1000, 2000 and 5000 ppm were administered to 4 animals (2 males and 2 females). At 500 and 1000 ppm, no overt signs of toxicity were noted. At 2000 ppm, a moderate to marked decrease in body weight and food consumption was seen in the male. At 5000 ppm, a slight to moderate decrease in body weight and food consumption was observed in the male and female. No treatment-related laboratory or histopathological changes were noted. Taking into consideration these findings, one male and one female were then treated daily with the read across substance at 2000 ppm (i.e., equivalent to 43 and 53 mg/kg bw/day (21.5 and 26.4 mg a.i./kg bw/day) in males and females respectively) for 2 weeks (Phase II). Under the study conditions, reduced food consumption was recorded throughout the treatment period. A slight decrease in protein, albumin and triglyceride levels and alkaline phosphatase activity was noted as well. Consequently, the dose levels of 250, 500 and 1000 ppm of active read across substance were chosen for the 28-day repeated dose toxicity study in beagle dogs (Gaou, 2004).

Main study: A 28-day study was conducted to determine the repeated dose oral toxicity of the read across substance, C12 -16 ADBAC (purity 49.9%) in Beagle dogs, according to OECD Guideline 409, in compliance with GLP. In this study, the read across substance was administered to 2 dogs per sex per group, at dietary doses of 0, 500, 1000 and 2000 ppm, corresponding to approximately 0, 250, 500 and 1000 ppm a.i., respectively. The homogeneity and stability of the read across substance under the administration conditions was checked before treatment start. Concentrations were measured in each dietary admixture in Week 1 and 4. No treatment-related effects were observed up to the highest tested dose. Under the conditions of the study, the 28-day NOEL for systemic effects in Beagle dogs was established at the highest tested dose of 1000 ppm a.i. (i.e., equivalent to a mean actual dose of 36.70 mg a.i./kg bw/day) (Gaou, 2006).

Study 4: A 90-day study was conducted to determine the repeated dose oral toxicity of the read across substance, C12-16 ADBAC (49.6% active in water) in Beagle dogs according to OECD Guideline 409, in compliance with GLP. The read across substance was administered to four animals per sex per dose group at dietary doses of 0, 500, 1500 and 3000 ppm (i.e., equivalent to 0, 250, 750 or 1500 ppm a.i.). From Week 8, the concentration of read across substance was reduced to 2500 ppm (i.e., 1250 ppm a.i.) in the high dose female group due to low food intake and reduced body weight among these animals (up to 20%). The mean achieved dosage of active substance, based on food consumption and body weight, were 0, 8, 25 and 50 mg a.i./kg bw/day for males and 0, 9, 26 and 45 mg a.i./kg bw/day for females. One out of 4 female dogs in the high dose group (1500 ppm a.i.) showed emaciated appearance and soft faeces. No other clinical signs were attributed to treatment with the read across substance.Themean body weight gain were recorded to be similar to the control females following reduction of the high dose group to 1250 ppm a.i. Consequently, the prior effects on body weights at 1500 ppm a.i. were considered to be due to reduced palatability. Also, slightly lower clinical chemistry parameters (i.e., mean protein and cholesterol levels) were noted in females from the high-dose group when given 1500 ppm a.i., consistently with the decrease of food intake. These differences were no longer observed at the end of the treatment period and after dose reduction to 1250 ppm a.i. Under the study conditions, the 90-d NOAEL for systemic effects in Beagle dogs was established at the highest adjusted test dose of 1250 ppm (i.e., equivalent 45 mg a.i./kg bw/day, respectively) (Guillaumat, 2006).

Chronic toxicity studies:

Study 1: A study was conducted to determine the repeated dose oral toxicity study of the read across substance, C12 -16 ADBAC (49.2 - 49.9% active in water) according to OECD Guideline 453, in compliance with GLP. This experiment evaluated the chronic toxicity and carcinogenic potential of the test substance in a combined study. The read across substance was administered daily to Sprague-Dawley rats by dietary admixture at the concentrations of 1000, 2000 and 4000 ppm (equivalent to 500, 1000 and 2000 ppm a.i.) for 52 weeks (toxicology sub-group) (equivalent to 28, 56 or 109 mg a.i./kg bw/d for males and to 33, 65 or 133 mg a.i./kg bw/d for females, respectively) and for 104 weeks (carcinogenicity sub-group) (corresponding to 24, 48 or 97 mg a.i./kg bw/d for males and 29, 58 or 119 mg a.i./kg bw/d for females). The read across substance did not induce any treatment-related mortality or clinical signs when administered daily for 52 or 104 weeks. At 4000 ppm, the mean body weight and body weight gain of the males of the toxicology sub-group and of the males and females of the carcinogenicity sub-group were lower than that of the controls, correlating with slightly lower mean food consumption. There were no significant differences in haematological, biochemical and/or urinalysis parameters at any dose-level for animals of either sub-group, compared with controls. There were no macroscopic or microscopic findings attributable to the read across substance at any dose levels. Under the study conditions, due to the observed lower body weight gain at 4000 ppm substance (significant only in males of 52-week chronic study part; significant in males and females in 104-week carcinogenicity group), the NOEL was established at 2000 ppm (equivalent to 56 and 65 mg a.i./kg bw/d for males and females for chronic groups or 48 and 58 mg a.i./kg bw/d in males and females for carcinogenicity groups) (Appelqvist, 2007).

Study 2: A study was conducted to determine the repeated dose oral toxicity of the read across substance (81.09% in aqueous/ethanol solution) according to OECD Guideline 453 and US EPA OPPTS 870.4300, in compliance with GLP. This two-year combined dietary toxicity and carcinogenicity study was conducted in Sprague-Dawley CD rats. The read across substance was administered rats (60/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm read across substance (equivalent to mean intake levels of 0, 13, 44, and 88 mg/kg bw/d in males and 0, 17, 57 and 116 mg/kg bw/d in females) in the diet daily for 104 weeks. There were two control groups of 60/sex/group each. The animals were observed twice daily and body weights and clinical findings were recorded periodically. Clinical pathology measurements (haematology, clinical chemistry and urinalysis) were made at 6, 12, 18 and 24 months. At termination, a thorough post-mortem examination was conducted on all animals. Histopathology was conducted on a full set of tissues and organs from all animals in the control and high dose groups as well as on selected organs from animals in the low and mid-dose groups. An increased incidence of loose faeces in male rats was observed which was considered to be potentially treatment-related, however, was not of biological significance. A reduction in mean absolute bodyweights and food consumption was observed in males and females in the high dose group. No other treatment related effects were observed for clinical pathology, organ weights, gross and histopathology or ophthalmology. Based on the results of the study, the NOAEL for chronic toxicity was determined to be 1000 ppm in diet (44 mg/kg bw/d for males and 57 mg/kg bw/d for females, equivalent to 35.64 and 46.17 mg a.i./kg bw/d respectively) (Gill, 1991).

As per the Biocides assessment report on C12-16 ADBAC, which was published by the Italian authorities in June 2015, reported the above studies and stated that “the effects on which the NOEL derivation could have been based, independently on the species tested, was the reduction in body weight and body weight gain, consistent with decreased food consumption (US ISC; EQC). It was concluded that all effects could be attributed to local gastrointestinal irritaton/corrosion and consequent reduced food intake without observing any primary systemic effect. Therefore, the derivation of a NOAEL for systemic effects was deemed inappropriate."

Therefore, in line with the 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, derivation of systemic NOAEL andDNEL has been considered to be non-relevant and only a qualitative local risk assessment has been conducted for the test substance. 

Inhalation:

The substance is considered to have a low vapour pressure (VP = 0.0058 Pa at 25°C, based on read across), which is below the cut-off of 0.01 Pa set for defining low volatility substances, as per the ECHA Guidance R.7a. Therefore, due to its solid physical state and low VP, the test substance is unlikely that it will form inhalable dust, mist or fumes when handled and used in solid form. In case inhalable forms of the substance (either pure or in aqueous solutions) are created under particular conditions (e.g., spraying, elevated temperature/pressure), appropriate risk management measures (due to corrosive nature of the test substance) such as closed systems, exhaust ventilation or wearing of respirators are implemented to control exposure. Under such conditions, the risk to humans following inhalation exposure can be considered minimal and further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. Nevertheless, a qualitative risk assessment has been carried out for this route, due to the corrosive nature of the test substance and the fact that the available repeated dose oral studies with the read across substance did not show any primary systemic effects; all observed effects were attributed to local gastrointestinal irritation/corrosion and consequent reduced food intake.

Dermal:

A repeated dose dermal toxicity study for the test substance is not required because the endpoint can be assessed based on the available sub-chronic oral studies with the read across substance, C12-16 ADBAC, which indicated that the main critical effects were due to the corrosive properties of the substance. Further, given the corrosive nature of the test substance together with the fact that the toxicokinetic assessment did not indicate higher absorption potential for the dermal route, any further testing on animals may be omitted due to animal welfare reasons, in accordance with Annex XI (1.2) of the REACH regulation. Nevertheless, a qualitative risk assessment has been carried out for this route, due to the corrosive nature of the test substance.

Based on the observed effects and the available NOAELs and LOAELs from the repeated dose studies, the test substance does not warrant a classification for STOT RE according to the EU CLP criteria (Regulation 1272/2008/EC).