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Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Objective of study:
absorption
distribution
excretion
Qualifier:
no guideline followed
Principles of method if other than guideline:
The absorption, distribution and excretion of test substance was determined by performing in vivo study in rats. Female Sprague-Dawley rats were starved for 24 h and then given [14C] test substance (0.8 mg/kg) as an aqueous solution by gastric intubation. The distribution of the 14C-labelled test substance was studied using collected samples of blood, organs and tissues by radioassay. The excretion of 14C-labelled test substance was studied using collected samples of urine, faeces and bile by thin layer chromatography and radioassay. [14C] toluene was used as internal standard. The results were reported as radioactivity (% of administered dose of test substance).
GLP compliance:
no
Remarks:
Study from Pre-GLP period
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Route of administration:
other: Oral - Gastric intubation
Vehicle:
water
Dose / conc.:
0.8 other: mg/kg of [14C] C16 TMAB in 4.0 mL/kg bw
No. of animals per sex per dose:
Distribution study: 6 groups of 3-5 animals
Excretion study: 4 animals
Bile collection: 3 animals
Control animals:
no
Details on dosing and sampling:
- Specific activity of test substance: 17.6 µCi/mg
- Volume applied: 4.0 mL/kg bw
- Exposure period: Up to 96 h
- Sampling time
Distribution study: 2, 4, 8, 24, 72, and 96 h
Excretion study: Urine and faeces were collected at 4 h intervals for 3 d.
Bile: 2 h intervals for 12 h
- Samples: Blood, urine, faeces, bile, organs (liver, kidney, spleen, skeletal muscle, hart, lungs)

Details:
Distribution studies
Female Sprague-Dawley rats (210-240 g) were starved for 24 h and then given [14C] CTAB (0.8 mg/kg) as an aqueous solution by gastric intubation. The administered volume was 4 mL/kg body weight. Blood samples were taken from the tail under mild ether anesthesia. The animals were killed 2, 4, 8, 24, 48, 72 and 96 h after intubation. In experiments lasting for more than 24 h the animals were provided with food and water ad lib. 8 h after intubation. Tissue samples weighing 100—200 mg were cut in duplicate from the liver, kidneys, spleen, heart, lungs and hind leg (gastrocnemius muscle) for radioassay. In animals killed 8 h after gastric intubation, the gastro-intestinal tract was removed and divided into four parts, namely the stomach, the proximal and distal halves of the small intestine and the caecum together with the colon. The contents of the different parts of the gastro-intestinal tract were collected by rinsing with saline.
 
Excretion studies
For the study of the excretion of 14C-labelled compounds, similarly treated animals were kept in metabolism cages permitting the separate collection of urine and faeces, which were removed at 4 h intervals for a period of 3 days and kept refrigerated until examined. Faeces were homogenized in ethanol and aliquots were taken for radioassay. Expired CO2 was trapped in 10% NaOH at 4 h intervals during day 1 after intubation. Saturated BaCl2 was added and the precipitated BaC03 was filtered off and dried, aliquots being taken for radioactivity determinations.
 
Bile collections
Biliary excretion was studied by means of a polyethylene cannula inserted into the common bile duct of rats anaesthetized with sodium pentobarbitone (40 mg/kg, ip). When bile flowed freely, the abdominal incision was closed and an aqueous solution of (0.8 mg/kg) was administered by gastric intubation. The bile was collected for 12 h at 2 h intervals and the samples were refrigerated until examined.
Type:
distribution
Results:
80% in GIT; 2% found in bile, 1.2% in urine, organs < 0.1%
Type:
excretion
Results:
92% excreted in faeces and 1% in urine; no radioactivity was detected in expired CO2
Type:
absorption
Results:
Based on the radioactivity found in excreta, total approx. absorption could be considered to be 3.3% (poor absorption)
Details on absorption:
About 80% of the dose of radioactivity was found in the gastro-intestinal tract 8 h after administration, only small amounts were found in the blood plasma and about 2% of the administered radioactivity was excreted in the bile during the first 12 h after treatment. The low levels of radioactivity in the serum and bile, together with the large amounts of radioactivity found in the gastro-intestinal tract, indicated poor intestinal absorption of C16 TMAB.
Details on distribution in tissues:
Distribution of radioactivity
The distribution of radioactivity in the gastro-intestinal tract 8 h after oral administration of [14C] C16 TMAB A total of about 80% of the administered radioactivity was found in the gastro-intestinal tract, about 87% of this amount being in the gastro-intestinal contents. About 90% of the administered dose had left the stomach within 8 h. Only small amounts of radioactivity were found in tissues other than the gastro-intestinal tract. The level of radioactivity in all the organs examined exceeded that of the blood plasma, the peak level in which occurred 2-4 h after administration of the dose. The liver and kidneys showed the highest levels of radioactivity, the peak in these two organs occurring approximately 8 h after dosing. At that time, assuming an even distribution of radioactivity in the liver tissues, the liver contained about 0.8% of the administered radioactivity, but 4 days after the administration of [14C] C16 TMAB, only traces of radioactivity remained in the liver and kidneys. The amount of radioactivity found in the skeletal muscle and spleen was 5-10% of that found in the liver. In the heart and the lungs, the levels of radioactivity were about the same as those in skeletal muscle.
Details on excretion:
Excretion of radioactivity
About 2% of the administered dose was excreted in the bile during the first 12 h after administration of [14C] CTAB. There was thus no appreciable enterohepatic circulation of radioactivity. The low levels of radioactivity in the serum and bile, together with the large amounts of radioactivity found in the gastro-intestinal tract, indicated poor intestinal absorption of CTAB. Thin- layer chromatography of bile revealed five spots of radioactivity all with an RF value smaller than that of the standard. Cationic surfactants can form ion-pairs with organic anions and it has been suggested that quaternary ammonium compounds are transferred across the gut wall as neutral complexes by virtue of their combination with endogenous anions. To test whether such a complex could explain the spots found on the chromatograms, [14C] CTAB was incubated with bile collected from the animals before treatment. However, chromatography of these incubates showed only one radioactive spot, with an RF value similar to that of the standard. After 3 days, 92% of the administered dose of radioactivity had been excreted in the faeces and only in the urine. Thin-layer chromatography of ethanolic faecal extracts revealed three radioactive spots that with the same RF value as the [14C] CTAB standard accounted for about 85% of the radioactivity in the faeces collected during day 1, while the other two spots had RF values smaller than that of the [14C] CTAB standard. In the urine, the peak levels of radioactivity were found in samples collected 4-8 h after dosing. Thin-layer chromatograms or urine samples showed four radioactive spots, the RF values of which were in one case similar to and in the others smaller than the [14C] CTAB standard. Faecal homogenates and urine incubated with showed onlv one radioactive spot, with the same RF value as the standard. No radioactivity was found in the expired CO2, collected during day 1 after administration of [14C] CTAB.
Metabolites identified:
not measured

Table 1: Distribution radioactivity in the gastro-intestinal tract of rats 8 h after oral intubation of 0.8 mg [14C] CTAB/kg 

Region of gut

Radioactivity (% of administered dose*)

Stomach

10 ± 3.1£

Small intestine, proximal half

2.5 ± 0.3£

Small intestine, distal half

13.5 ± 1.5£

Caecum and colon

53.8 ± 2.4£

Complete gastro-intestinal tract: Total

79.9 ± 1.3£

Contents

69.8 ± 2.4

Wall

10.1 ± 2.3

Gastric emptying

90 ± 3.1

*Values represent means ± SEM for five rats

£ Wall plus contents

 

 

Table 2: Distribution of radioactivity in urine and faeces after oral intubation of 0.8 mg [14C] CTAB/kg

Radioactivity

(% of administered dose*)

Day

Faeces

Urine

Faeces + Urine

1

89.10± 2.23

1.06± 0.11

90.16±1.98

2

2.83± 1.20

0.13± 0.02

2.96±1.06

3

0.56± 0.17

0.03± 0.02

0.59±0.15

Total

92.49± 2.06

1.22± 0.10

93.71±2.15

*Values represent means SEM for four rats

Conclusions:
Based on the results of the read across study, the test substance can be assumed to have very low absorption (i.e., <10%), distributed mainly in GIT and excreted in faeces.
Executive summary:

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

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From January 06, 2004 to November 17, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
- Study was conducted according to the OECD 417 and in compliance with GLP with some acceptable deviations. However, the dermal application part suffered from design limitations, allowing for oral uptake from the skin after the 6h exposure, and therefore invalidating the results obtained for dermal uptake.
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Objective of study:
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
see any other information on materials and methods incl. tables section
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
-Species: Sprague-Dawley rats
-Strain: Crl CD® (SD) IGS BR
-Source: Charles River Laboratories France, L’Arbresle, France. Caesarean Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
-Sex: 60 males and 60 females.
-Age/weight at study initiation: Young adults approximately 7 week old; for the bile collection group, animals were around 10 week old.
-Number of animals per group: Kinetics (5 groups): 9 males and 9 females (3/group/time/sex); Excretion balance: (3 groups): 5 males and 5 females
Bile collection (1 group): 4 males and 4 females
-Control animals: Yes: For the purposes of pre-dose sample analysis, plasma, blood and tissues will be collected from at least one untreated supplementary animal/sex using the above mentioned procedures.

Route of administration:
other: Oral by gavage and dermal
Vehicle:
water
Details on exposure:
-Specific activity of test substance 2.15 MBq/mg test item (58.2 µCi/mg)
-Volume applied: Oral: 10 mL/kg; Dermal: 1.5 mL/kg bw on approximately 10% body surface area (approximately 12 µL/cm2)
-Size of test site: Dermal: approximately 10% body surface area: 25 cm2 for 200 g rat, 30 cm2 for 250 g rat.
-Exposure period: Dermal: Treated area washed after 6 h.
-Sampling time: PK - oral: (3 animals/sex/group) post-gavage
⋅ first sampling set: 0.5, 4 and 24 h,
⋅ second sampling: 1, 8 and 48 h,
⋅ third sampling set: 2, 72 and 96 h.
PK - dermal (after initiation of skin contact):
⋅ first sampling set: 3, 8 and 24 h,
⋅ second sampling: 6, 10 and 48 h,
⋅ third sampling set: 7, 16 and 72 h.
MB oral and dermal:
Urine and faeces: 24 h before radioactive treatment, and during the periods 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 h after the radioactive gavage/dermal application.
Bile collection: 0-3, 3-6, 6-12 and 12-24 h post gavage.

-Samples: Blood/plasma, urine, faeces, bile, exhaled air, organs, carcass, skin with substance not removable, liquid used for washing the skin, protective appliances.

- Preparation of the site: The hair of the administration site was clipped (using electric clippers) the day before dosing.
Duration and frequency of treatment / exposure:
6 h
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
Males and females: Single and repeated oral low dose level: 50 mg/kg bw (for PK TD, MB and bile (single exposure)); high dose oral: 200 mg/kg bw (PK and TD); single dermal low dose: 1.5 mg/kg bw (for PK, TD and MB); high dose dermal: 15 mg/kg bw (for PK and TD).
PK= Pharmacokinetics; TK= Tissue distribution; MB = Mass balance
No. of animals per sex per dose:
60 males and 60 females with the below split:
Kinetics (5 groups): 9 males & 9 females (3 /group/time/sex)
Excretion balance: (3 groups): 5 males & 5 females
Bile collection (1 group): 4 males & 4 females
Control animals:
yes, concurrent no treatment
Details on study design:
For details kindly refes to the attached background material section of the IUCLID.
Details on dosing and sampling:
See 'Any other information on materials and methods incl. tables'.
Type:
other: Absorption, distribution and excretion
Details on absorption:
Dose: Radiochemical purity (99.5%) is sufficient (>98% ; OECD guidance), and formulated material was relevant in relation to possible human exposure: Concentrations for dermal application were 0.1 and 1% for 6h. This is based on already avaibale information on irritancy levels of the test substance: 4h, 5% on rabbit skin is mildly irritating; 24h occluded patches with 0.1% concentrations in water produced a well defined erythema and very slight oedema.

The oral high dose of 200 mg a.i./kg bw could possibly lead to some toxic effects, as the acute LD50 by gavage is between 250 and 450 mg a.i./kg bw.

Recovery: Total recovery for oral groups were suffficient with around 100% except for males of repeated dose group resulting to 95.3% recovery. For dermal groups, total recovery was slightly low (87.2% males and 91.0% females). However, it should be remarked that probably the total radioactivity in the carcass samples with an avarage of about 5% was too low, as for all 4 animals for which no seperate internal organs were measured, total carcass levels were about 10%, compared to 1 to 3% for the 6 animals for which radioactivity in individual oragns were measured.

Oral absorption:
Most of the radioactivity were excreted via the faeces. About 3-4% left via urine, except for the males in the repeated dose groups showing a mean value of 8.31%. However, soft faeces were also observed in this group during the days of collection, possibly causing contamination of urine with faeces.
Also corresponding with high cage wash values. Elimination was quick, with 70-80% excreted within the first 24h. No radioactivity was left in the carcass. Bile examinations show that 4.58% (males) and 3.75% (females) of the total dose was recovered in the bile. Elimination was quick, with 25-30% already passed out in the bile in the 0-3 h period. The mean plasma and blood levels for males and females remained below quantifiable limits at all time points, except in the 50 mg/kg dose group for 0.5 to 2h time points for plasma (161 and 251 ng -eq/g for males at 1 and 2h respectively, and 109 ng-eq/g at 0.5 h, 212 ng-eq/g at 1h and 192 ng-eq/g at 2 h for females), and in blood only the 1 h time point in females (173 ng-eq/g). No plasma or blood levels could be determined for the 200 mg/kg dose group or the repeated 50 mg/kg dose group.

Following single oral gavage at a nominal dose-level of 50 mg/kg bw to rats of group 1, the mean radioactivity levels were below quantifiable limits in all tissues/organs at all time-points, except for the intestines and liver. Specifically, levels for males/females were 23.3/23.2% of the dose for the intestines and 0.087/0.039% of the dose for the liver at the 24h time-point. Levels decreased over time, and were all non-quantifiable by 168h.

Following single oral gavage at a nominal dose level of 200 mg/kg bw to rats of group 2, the mean radioactivity levels were above quantifiable limits in approximately half the analysed tissues and organs at 24 h. Specifically, high levels were present in the intestines (62.2/71.5% of the dose) for males/females, and trace levels were present in the abdominal fat, heart, kidneys, liver, lungs, lymph nodes and or pancreas (range 0.004 to 0.24% of the dose). As previous, levels decreased over time, and were all non-quantifiable by 96h with the exception of the intestines.

Following repeated oral gavage at a nominal dose-level of 50 mg/kg bw to rats of group 5, the mean radioactivity levels were below quantifiable limits in all tissues/organs, except for the intestines (e.g. levels for males/females of 15.9/36.9% of the dose at 24h) and the liver in males (0.072% of the dose at 24 h). Levels decreased over time and were non-quantifiable by 168h.

Dermal absorption:
The minimal percutaneous absorption (Faeces, urine and intestines) seemed to amount to 46.4% for males, and 47.4% for females. The maximum systemic absorption (faeces, urine, carcass and skin site) was 50.0% and 50.1% for males and females respectively. The data indicate that the skin application site is a reservoir for absorbed radioactivity in the animals. As the dermal application site was not protected from grooming after the 6h exposure period, test substance remained available on the skin for subsequent oral uptake from grooming.

Test substance was uniformly distributed in the stratum corneum. Cross-contamination to adjacent skin was observed. The mean plasma and blood levels for males and females for 1.5 mg/kg dose group remained below quantifiable limits at all time points, except for the 7 and 8 h time-points for blood (levels for males/females of 3.52/4.40 and 2.67/3.26 ng-eq/g, respectively). For the 15 mg/kg bw dose group only the 8 (levels of 70.2/68.6 ng-eq/g for males/females) and 24 (levels of 62.3/55.0 ng-eq/g for males/females) h time-points resulted in values above the quantifiable limits.

Following single dermal application at a nominal dose-level of 1.5 mg/kg to rats of group 3 and 15 mg/kg to rats of group 4, the mean radioactivity levels were below quantifiable limits in all tissues/organs at all time-points except for the intestines, "stripped" skin fiom the application site and adjacent site. Trace levels were found at 24 and 48h time-points in the eyes of the 1.5 mg/kg bw dose group.
Details on distribution in tissues:
See above for details.
Metabolites identified:
no
Details on metabolites:
The radioactivity excreted in the urine was not associated with the parent compound, but metabolites formed were not identified.

Toxic effects and clinical signs:

No test substance-related mortality or morbidity were observed during the study. The only death observed (Male D28768, group 9, single oral dose at 50 mg/kg bw), was an isolated incident and not seen at the higher dose-level of 200 mg/kg bw, was considered to be due to complications following the bile duct cannulation. In terms of clinical signs, ptyalism was seen in 1/9 males and 1/9 females of group 5 from Day 6 (repeat oral dose of 50 mg/kg bw) and soft faeces were noted in 4/5 males of group 8 from Day 6 (also, repeat oral dose of 50 mg/kg bw). As the ptyalism was of low incidence and is often seen in rats treated orally, this was not considered to be test substance-related. In contrast, as of high incidence, the soft faeces were considered to be due to the repeated test substance treatment. No dermal irritation occurred.

Dermal irritation: No effects

Recover of labelled compound (Mass balance (Group 6, 7 & 8)):

Oral:Following single or repeated oral gavage of the isotopic test item mixture at a nominal 50 mgkg/day to rats, the mean (+/- standard deviation) total cumulative excretion of the radioactive dose for males/females over a 168-hour period in urine, feces and cage wash was complete at 100.3 +/-2.2/101.4 +/- 7.2% for the single dose group 6 and 95.3 +/- 5.7/101.2 +/- 2.1% for the repeated dose group 8.

Dermal:Overall, the absolute total recovery of radioactivity was slightly low at 87.2 ± 2.9% for males and 91.0 ± 1.8% for females; in view of the homogeneity of the data, the low absolute total recovery of radioactivity was of unknown cause, but may have been due to poor estimation of the applied radioactive dose or poor recovery of radioactivity from the site wash materials. Losses of radioactivity as14CO2are considered to be unlikely in view of the correct mass balance obtained for the oralroute.

Conclusion:

-Oral application: 
Following single and/or repeated oral gavage at 50 and 200 mg/kg bw/day, 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 about 8% (urine and bile fractions); this was eliminated rapidly, essentially within a 48 to 72h period. The vast majority of the oral dose was excreted rapidly in the faeces. At the high oral dose-level only, quantifiable levels of radioactivity were found in some central organs at 8h post-dosing; otherwise, the vast majority of the dose was confined to the intestine and levels decreased over time. Only 0.62 to 8.15% of the oral
dose was eliminated in the bile in a 24h period.

-Dermal application:
Following single dermal application at 1.5 and 15 mg/kg bw, 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 48h period, suggesting that the dermal dose was highly absorbed via the skin. However, as the test site was not protected with an Elizabethan collar during the main part of the collection
period (the collar was worn during the 6h exposure period only), this may have been due to the animal licking the test site. This is also supported with the finding that after oral dosing only about 4% was excreted via bile back to intestines, and 4% excreted via urine. If similar routes of excretion are expected for dermal absorbed doses, it would not be possible to find levels of 50% of applied doses in intestines with only 2% excreted via urine. This indicates that about 50% of the dermal applied dose was taken up orally after all, which following the same oral kinetics 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 intestine 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 essentially decreased over time.

- All data showed low inter-animal variability. In addition, there was no evidence of gender differences.

Conclusions:
Under the conditions of the study 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.
Executive summary:

A study was conducted to determine the basic toxicokinetics of the read across substance, C12-16 ADBAC (49.9% active in water, 99.4% radiolabelled purity), according to OECD Guideline 417, in compliance with GLP. In this study, Sprague-Dawley rats were treated with single and repeated oral doses (50 or 200 mg/kg bw) as well as a single dermal dose (1.5 or 15 mg/kg bw) of the radiolabelled read across substance. Following single and/or repeated oral doses, the plasma, blood and organ radioactivity levels were essentially non-quantifiable, indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was around 8% (urine and bile fractions). This was eliminated rapidly, essentially within a 48 to 72 h period. The majority of the oral dose was excreted in the faeces. At the high oral dose level only, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines, where their levels decreased over time and were all non-quantifiable by 168 h (i.e., 7 d). Only about 4% of the oral dose was eliminated in the bile in a 24 h period, of which about 30% during the first 3 h. 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 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. 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 conditions of the study and following oral administration the read across substance was found to have limited absorption (ca. 10%), low distribution (below quantification limits within 4-7 d) and majorly excreted via faeces (ca. 80%). 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).

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Objective of study:
absorption
distribution
excretion
metabolism
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C-labelled test substance
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
- Source: Charles River
- Age at study initiation: 6 weeks
- Weight at study initiation: Average body weight: 268.6 g (male) and 167.0 g (female)

Details on exposure:
Preparation of dosing solutions:
Preliminary experiments: Oral gavage – single low dose
Experiment 1: Oral gavage – single low dose
Experiment 2: Dietary – repeated low dose
Experiment 3: Oral gavage – single high dose
Experiment 4: Intravenous


Vehicle:
Distilled water (Preliminary experiments and Experiments 1, 3 and 4)
Rodent diet/distilled water (Experiment 2)

Concentration in vehicle:
Preliminary experiments and Experiment 1: 1.0 mg/mL
Experiment 2: 100 ppm in diet for 14 d/1 mg/mL in water single oral dose
Experiment 3: 5.0 mg/mL
Experiment 4: 4 mg/mL


Homogeneicity and stability of the test material: Stable
Duration and frequency of treatment / exposure:
Preliminary experiments: Oral gavage – single low dose
Experiment 1: Oral gavage – single low dose
Experiment 2: Dietary – repeated low dose for 14 d
Experiment 3: Oral gavage – single high dose
Experiment 4: Intravenous
Dose / conc.:
10 mg/kg bw/day
Remarks:
Preliminary experiments and Experiment 1 and Experiment 4
Dose / conc.:
100 ppm
Remarks:
Experiment 2: 100 ppm non-radiolabelled substance for 14 d, followed by 10 mg/kg radiolabelled.
Dose / conc.:
50 mg/kg bw/day
Remarks:
Experiment 3
No. of animals per sex per dose:
Preliminary experiments: 2 per sex per group (Total 8 animals)
Main experiments:5 per sex per group (Total 40 animals)
Control animals:
no
Details on dosing and sampling:
Pharmacokinetic study (Absorption, distribution, excretion):
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes
- Time and frequency of sampling: Urine, faeces and urine/feces separator washing samples were collected at the following time intervals: 0-4, 4-8, 8-12, 12-24, 24-36, 36-48, 48-72, 72-96, 96-120, 120-144 and 144-168 h.

Metabolite characterisation studies:
- Tissues, urine and faeces were collected and analysed for radioactivity and faeces were analysed by TLC, HPLC and MS for metabolites and parent compound.



Preliminary studies:
Results in preliminary studies indicated that negligible amounts of 14CO2 (0.020%) were found in expired air, demonstrating that radiolabel was localized in a stable portion of the molecule
Details on absorption:
Following oral administration, 14C-ADBAC was rapidly absorbed, although in very limited amount as indicated by the low blood levels (<0.003% of the administered dose/ml) attained starting from 15 min after dosing and peaking between 3 and 8 h. Blood concentration declined to approximately 25% of the peak value within 24 h.

Based on data on urine excretion and tissue residues after oral administration, and indication from i.v. experiment, it can be expected that the test subsatnce absorption through the g.i. tract is about 20% (conclusion not included in the study report; as assessed by the Italian Rapporteur Member state in the biocide dossier).
Details on distribution in tissues:
Residual 14C in tissues was negligible (<1%) after administration of radiolabelled test substance 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.
Details on excretion:
Percent Recovery:

Experiment 1:
Males: 5.77% urine; 98.61% faeces
Female: 6.88% urine; 91.20% faeces
Total Recovery: 104.54 ± 5.29% - males; 98.11 ± 3.25% females

Experiment 2:
Males: 4.76% urine; 95.12% faeces
Female: 5.80% urine; 97.22% faeces
Total Recovery: 100.19 ± 4.94% - males; 103.1 ± 5.18% females

Experiment 3:
Males: 7.75 % urine; 90.03% faeces
Female: 6.95% urine; 87.48% faeces
Total Recovery: 98.36 ± 2.42% - males; 94.58 ± 7.57% females

Experiment 4:
Males: 30.63% urine; 44.44% faeces
Female: 20.58% urine; 55.09% faeces
Total Recovery: 108.43 ± 5.56% - males; 111.45 ± 3.96% females

Remarks:
- 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 is not possible to conclude if this radioactivity accounts exclusively for 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.
- Little or no gender or dose-dependent differences were observed in the excretion patterns nor in rats receiving a single or repeated dose.
- Based on the 5-8% of the test substance administered dose eliminated via urine and tissue residues (less than 1% of the administered dose 7 days after single and repeated oral dosing), it can be expected that the test substance absorption through the g.i. tract is about 10% (ECHA biocides assessment report, 2015).
- 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 (ECHA biocides assessment report, 2015)
Metabolites identified:
yes
Details on metabolites:
Over 50% of the faecal radioactivity was unchanged parent compound. Four major metabolites were identified, as oxidation products of the two decyl side chains to hydroxy and hydroxyketo derivatives. The only metabolism which occurred involved oxidation of the two decyl side chains to hydroxy and hydroxyketo derivatives. All were more polar and presumed less toxic than the parent compound. It is predicted that there is no major metabolite greater than 10% of the dosed radioactivity.

All rats receiving the test substance i.v. experienced clear signs of toxicity, including death, and all but one showed red urine during the first 4 h after the treatment.

Conclusions:
Under the conditions of the read across study, the test substance was found to have limited absorption (ca. 10%), 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  Repeated dosing did not alter the uptake, distribution or metabolism of test substance.
Executive summary:

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

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From August 18, 2005 to August 17, 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Species:
human
Details on test animals and environmental conditions:
Human skin membranes, in vitro
Type of coverage:
other: Automated flow-through diffusion cell system
Vehicle:
water
Doses:
0.03% (w/w) and 0.3% (w/w), in water
No. of animals per group:
In vitro study
Details on in vitro test system (if applicable):
Skin preparation:
- Source of skin: Eight samples from Plastic Surgery unit, St. Johns Hospital, West Lothian NHS Trust, Livingston, UK
- Ethical approval if human skin: Yes
- Type of skin: Full-thickness human skin (1 upper arm, 2 abdomen and 5 breast)
- Preparative technique: Skin was transferred to Charles River Laboratories on ice and cleaned of subcutaneous fat and connective tissue using scalpel. Skin was washed in cold running tap water and dried using tissue paper. Each sample was then cut into smaller pieces, wrapped in aluminium foil, put into self sealing plastic bags and stored at -20°C until required. Split-thickness membranes were prepared by pinning the full thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200-400 µm depth using a Zimmer electric dermatome.

Principles of assay:
- Diffusion cell: Automated flow-through diffusion cell system (Scott/Dick, University of Newcastle-upon-Tyne, UK)
- Receptor fluid: Tissue culture medium containing approximately 5% (w/v) bovine serum albumin, 1% (w/v) streptomycin (approximately 0.1 mg/mL), and penicillin G (approximately 100 units/mL)
- Solubility of test substance in receptor fluid:
- Flow-through system: Automated
- Test temperature: The mean temperature 19°C (SD=0.8°C)
- Humidity: The mean relative humidity 35% (SD=0.6%)
Total recovery:
Refer to Table-1
Key result
Dose:
0.03%
Parameter:
percentage
Absorption:
0.05 %
Remarks on result:
other: 24 h
Remarks:
96.80% was not absorbed
Key result
Dose:
0.3%
Parameter:
percentage
Absorption:
0.03 %
Remarks on result:
other: 24 h
Remarks:
94.68% was not absorbed

- Low dose (0.030%):

The mean mass balance was 99.03% of the applied dose (3.09 μg equiv./cm2). The mean dislodgeable dose was 60.53% of the applied dose (1.89 μg equiv./cm2). The mean total unabsorbed dose was 96.80% of the applied dose (3.02 μg equiv./cm2). This consisted of the dislodgeable dose, unexposed skin (0.02%) and the radioactivity associated with the stratum corneum (36.25%). The stratum corneum acted as a good barrier to the test substance as the bulk of the radioactivity (30.26%) was recovered in the outermost 5 tape strips (tape strips 1-5). Considerably less radioactivity was recovered with each of the subsequent 3 groups of tape strips (3.20%, 1.82% and 0.97% in tape strips 6-10, 11-15 and 16-20 respectively), suggesting that the test substance would be sloughed off with the skin in the future. The absorbed dose (0.05%, or <0.01 μg equiv./cm2) was the sum of the receptor fluid (0.05%) and the receptor rinse (<0.01%). Dermal delivery (2.22%, or 0.07 μg equiv./cm2) was the sum of the absorbed dose and the exposed skin (2.18%). There was no apparent lag time and the fluxes ranges from 0.03 to 0.12 ng equiv./cm2/h over the 1 to 24-h exposure period.

 

- High dose (0.300%):

The mean mass balance was 96.84% of the applied dose (29.91 μg equiv./cm2). The dislodgeable dose was 77.87% of the applied dose (24.05 μg equiv./cm2). The mean total unabsorbed dose was 94.68% of the applied dose (29.24 μg equiv./cm2). This consisted of the dislodgeable dose, unexposed skin (0.17%) and the radioactivity associated with the stratum corneum (16.64%). The stratum corneum acted as a good barrier to the test item as the bulk of the radioactivity (10.86%) was recovered in the outermost 5 tape strips (tape strips 1-5). Considerably less radioactivity was recovered with each of the subsequent 3 groups of tape strips (3.11%, 1.71% and 0.96% in tape strips 6-10, 11-15 and 16-20, respectively), again suggesting that the test item would be sloughed off with the skin in the future. The absorbed dose (0.03%, or 0.01 μg equiv./cm2) was the sum of the receptor fluid (0.03%) and the receptor rinse (<0.01%). Dermal delivery (2.16%, or 0.67 μg equiv./cm2) was the sum of the absorbed dose and the exposed skin (2.13%). There was no apparent lag time. There was no apparent lag time and the fluxes ranges from 0.22 to 0.74 ng equiv./cm2/h over the 1 to 24-h exposure period.

Table 1. Summary of recoveries after 24h

Test Preparation

Low Concentration

High Concentration

Target test substance concentration (%, w/w)

0.03

0.30

Test substance concentration by Radioactivity (%, w/w)

0.031

0.306

Test preparation application rate (mg/cm2)a

10.01

10.09

Test substance application Rate (μg equiv./cm2)

3.12

30.87

Dislodgeable Dose (% Applied Dose)

60.53

77.87

Unabsorbed Dose (% Applied Dose)

96.80

94.68

Absorbed Dose (% Applied Dose)

0.05

0.03

Dermal Delivery (% Applied Dose)

2.22

2.16

Mass Balance (% Applied Dose)

99.03

96.84

Dislodgeable Dose (μg equiv./cm2)

1.89

24.05

Unabsorbed Dose (μg equiv./cm2)

3.02

29.24

Absorbed Dose (μg equiv./cm2)

<0.01

0.01

Dermal Delivery (μg equiv./cm2)

0.07

0.67

Mass Balance (μg equiv./cm2)

3.09

29.91

a mg of test preparation per cm of skin

 

Conclusions:
Under the conditions of the read across study, the mean absorbed dose and mean dermal deliveries were determined to be 0.05% and 2.22% of the applied dose for the low concentration test preparation and 0.03% and 2.16% of the applied dose for the high concentration test preparation, 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, indicating low absorption potential.
Executive summary:

An in vitro study was conducted to determine the rate and extent of dermal absorption of the read across substance, C12-16 ADBAC (80.5% active; >99% radiolabelled purity), according to OECD Guideline 428, in compliance with GLP. The study was conducted with radiolabelled read across substance at 0.03% and 0.3% concentrations, which was topically applied over split-thickness human skin membranes mounted into flow-through diffusion cells. Receptor fluid was pumped underneath the skin at a flow rate of 1.5 mL/hour. The skin surface temperature was maintained at approximately 32°C. A barrier integrity test using tritiated water was performed and any skin sample exhibiting a permeability coefficient (kp) greater than 2.5 x 10-3 cm/h was excluded from subsequent absorption measurements. The 14C- radiolabelled read across substance was applied at an application rate of 10 mg/cm2. Absorption was assessed by collecting receptor fluid in hourly intervals from 0-6 h post dose and then in 2-hourly intervals from 6-24 h post dose. At 24 h post dose, the exposure was terminated by washing and drying the skin. The stratum corneum was then removed from the skin by 20 successive tape strips. All samples were analysed by liquid scintillation counting.Under the conditions of the study, the mean absorbed dose and mean dermal deliveries were determined to be 0.05% (0.01 ηg equiv. /cm2) and 2.22% (0.07 ηg equivalent/cm2) of the applied dose for the low concentration test preparation, respectively, and 0.03% (0.01 ηg equivalent /cm2) and 2.16% (0.67 ηg equivalent/cm2) of the applied dose for the high concentration test preparation, respectively. The stratum corneum acted as a barrier to absorption, with the mean total unabsorbed doses (recovered in skin wash, tissue swabs, pipette tips, cell wash, stratum corneum and unexposed skin) of 96.80 and 94.68% of the applied dose for the low and high concentration test preparations, respectively. The maximum fluxes for the low and high doses were 0.12 ηg equivalent /cm2/h and 0.74 ηg equivalent /cm2/h, respectively, at 2 h (Roper, 2006).

Endpoint:
dermal absorption in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The percutaneous absorption of the test substance was determined by performing in vivo cutaneous application study in rats. The 240 µL sample of a 3% solution [14C] test substance water was applied to the intact clipped skin of male Wistar rats under occlusive conditions. The samples of urine, faeces, rinsing, blood, removed treated skin, homogenized carcass and glass cap (occlusive dressing) were collected. The Radioactivity was determined (% of 14C administered dose) for samples to calculate the percutaneous absorption of test substance.

GLP compliance:
no
Remarks:
Study from Pre-GLP period
Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Male Wistar rats (200-230 g)
Type of coverage:
other: Light glass cap (fitted with small holes to avoid occlusive conditions and was glued to the skin with a special adhesive, care being taken to avoid contamination of the treated area with the adhesive)
Vehicle:
water
Duration of exposure:
5 and 15 minutes, 48 h
Doses:
0.5% 1% and 3% test substance in water
No. of animals per group:
1 group of 3 animals
Control animals:
no
Details on study design:
- Preparation of test site: Shaven skin
- Concentration of test substance: 3% concentration of [14C]-N-dodecyltrimethylammonium bromide
- Specific activity of test substance: Not indicated
- Volume applied: 240µL
- Size of test site: 8 cm² or other
- Exposure period: 2 days
- Sampling time: Blood: 0.5, 1, 5, 22, 26, 30, 46, 50; Urine & faeces: 24 and 48 h
- Samples: Blood, urine, faeces, carcass, skin with substance not removable
Signs and symptoms of toxicity:
not examined
Remarks:
Not indicated – unlikely with low TMAC dose applied
Dermal irritation:
not examined
Total recovery:
96.4 ± 7.09%
Key result
Time point:
72 h
Dose:
1%
Parameter:
percentage
Absorption:
ca. 0.59 %
Remarks on result:
other: study with radiolabelled test substance with rinsing
Key result
Time point:
48 h
Dose:
0.5%
Parameter:
percentage
Absorption:
ca. 0.093 %
Remarks on result:
other: study with radiolabelled hair rinse formulation of the test substance
Key result
Time point:
48 h
Dose:
3%
Parameter:
percentage
Absorption:
ca. 3.15 %
Remarks on result:
other: study with radiolabelled test substance without rinsing

Cutaneous application of [14C] test substance with rinsing

Application of 1% aqueous solution of [14C] test substance on the skin wih rinsing resulted in a very low percutaneous absorption of the surfactant. The total absorption was 0.59% of the applied radioactivity. Most of the amount absorbed was excreted in the urine. Within the first 24 h, 0.35% of the applied surfactant was excreted. Of the radioactivity applied, 13.2% remained on the skin after rinsing, demonstrating the relatively high affinity of the surfactant for skin.

Cutaneous application of [14C] test substance in a hair-rinse preparation

Application of [14C] test substance in a hair-rinse preparation (0.5% test substance) under conditions of normal use resulted in a marked decrease in the percutaneous absorption of the surfactant.The total absorption was 0.093% of the applied radioactivity. Only 0.016% of the amount applied was excreted in the first 24 h and 4.11% of the surfactant remained at the application site. No significant radioactivity was detected in the blood of the treated animals during the experiment (detection limit 10 ng surfactant/g blood).

Cutaneous application of [14C] test substance without rinsing

Application of a 3% solution of [14C] test substance on theskin without any rinsing resulted in percutaneous resorption of 3.15% of the applied radioactivity.In the experiments involving rinsing, the excretionof radioactivity wasalways lower on Day 2 than on Day 1, but in this experiment there was a marked increase in absorption on Day 2. The possibility cannot be excluded that the relatively long contact of the skin with the surfactant caused a slight but invisible damage to the skin, resulting in higher absorption rates. In two of the rats, the blood level of the surfactant was below the detection limit of 10 ppb (10 ng/g) for the first 5 h of cutaneous application of test substance.

Conclusions:
Under study conditions, percutaneous absorption of the [14C] read across substance was found to be 0.6% with rinsing and 3.15% without rinsing.
Executive summary:

A study was conducted to determine the percutaneous absorption of the radiolabelled read across substance, [C14] C12 TMAB (purity not specified), under occlusive conditions on rat skin. The read across substance was applied to the intact clipped skin of 3 rats under three scenarios: at 1% and 3% in aqueous solution followed by subsequent with and without rinsing respectively and 0.5% hair-rinse formulation of read across substance. Application in a cream hair-rinse preparation under user conditions resulted in the absorption of about 0.1% of the administered radioactivity after 48 h. No measurable radioactivity was present in the blood. However, application of the read across substance at 1% and 3% aqueous without subsequent rinsing solution gave a somewhat higher absorption (0.6% after 72 h and 3.15% after 48 h respectively), whereas, some radioactivity was found in the blood after application of the read across substance to the skin without subsequent rinsing. Overall the percutaneous absorption of the read across substance was low. Under study conditions, percutaneous absorption of the radiolabelled read across substance was found to be 0.6% with rinsing and 3.15% without rinsing (Bartnik 1979). Based on the results of the read across study, similar toxicokinetic profile is expected for the test substance.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From July to August, 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
Draft OECD TG 428: Percutaneous Absorption: in vitro Method (1994).
Deviations:
not specified
GLP compliance:
yes
Radiolabelling:
not specified
Species:
pig
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
Excised, dermatomed (1000 μm) pig skin (back and flank of castrated male pig) on a static diffusion cell
Type of coverage:
not specified
Vehicle:
water
Duration of exposure:
30 min (24 h; up to 72 h)
Doses:
The test substance (0.87 mg/cm2 of the active ingredient) was applied to the skin disks at an area dose of 25 mg/cm² (100 mg on 4 cm²) for an exposure period of 30 minutes and subsequently rinsed off with a neutral shampoo and water.
No. of animals per group:
6 skin samples
Details on study design:
Preparations of dermatomed pig skin measuring 1000 μm in thickness with stratum corneum, epidermis and parts of the dermis were used. Six skin samples were mounted in parallel in Teflon diffusion chambers which were continuously rinsed with receptor fluid (0.9% sodium chloride in distilled water).
Details on in vitro test system (if applicable):
6 skin samples (not stated whether they originated from one or more animals). Concentrations of the test substance in receptor fluid were determined at the start of the experiment (0 h) and after 16, 24, 40, 48, 64 and 72 h by HPLC/ESI/MS detection. In addition, the test substance was analysed in different skin layers and in the rinsing fluid in order to enable calculation of total recovery.
Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
At any of the different sampling times, small quantities of the test substance could be detected in the horny layer (1.25-14.25 μg/cm²) and in residual skin (0.75-7.25 μg/cm², corresponding to 0.086-0.83%, with a mean of 0.27±0.28%).
Total recovery:
The total recovery was about 108%.
Key result
Time point:
24 h
Dose:
3.5% in test substance in an emulsion on 0.87 mg/cm2 area of skin
Parameter:
amount
Absorption:
10.6 other: µg/cm2
Remarks on result:
other: 10.6 μg/cm2 (i.e., equivalent to 1.2% of the applied dose)

Table 1 : Amount of test substance in different part of test system:

Amount of the test substance in:

μg/cm2 (mean ± SD (range))

Receptor fluid

below detection limit (100 ppb)

Stratum corneum

6.1 ± 5.3 (1.25 - 14.25)

Dermis

2.3 ± 2.5 (0.75 - 7.25)

Rinsing solution

742 ± 38 (701.8 - 805.8)

Spatula/swabs/pipette

140 ± 51 (64.5 - 215.5)

Total recovery

891 ± 43 (842.0 - 960.5)

Since the horny layer of the skin has not been completely separated; the worst case situation should be considered. The amount found in the dermis (7.25 μg/cm² as upper level) was used for quantitative exposure assessment for rinse-off products.

The results expressed as a percentage were not considered relevant since the applied dose was clearly in excess. Therefore the percentages would provide an underestimation of the real dermal absorption. For the purposes of the risk assessment, a conservative value (limit of quantification, LOQ) for the receptor fluid may be taken as a worse case value with the assumption that the amounts in the receptor fluid were at the respective LOQ value. For the duration of 24 h, a mean value of about 3.3 μg/cm² (range 2.4-3.7 μg/cm²) can be calculated. Adding the amount of the test substance in dermis would result in10.6 μg/cm²as a worst case value.

Conclusions:
Based on the results of the read across study, the worst case dermal absorption value for risk assessment of the test substance was assumed to be 10.6 μg/cm2 (i.e., equivalent to 1.2% of the applied dose).
Executive summary:

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

Description of key information

Based on the available weight of evidence experimental studies, the test substance, C12-18 TMAC, is expected to have a poor absorption potential through oral and dermal routes and to be primarily excreted via faeces. Based on QSAR predictions and data on read across substance, it is likely to undergo aliphatic hydroxylation as the first metabolic reaction. Further, based on its ionic nature, molecular weight and key physico-chemical properties it is likely to have no or very bioaccumulation potential.



Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
10
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

ABSORPTION:

Oral absorption

Based on physicochemical properties:

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

The test substance, C12-18 TMAC is an alkyl trimethyl ammonium chloride (TMAC) is a cationic surfactant, which is a UVCB with majorly C12, C14, C16 and C18 alkyl chain lengths and molecular weight ranging from 207.79 to 346.04 g/mol. The purified form of the substance is a creamy white solid powder. It has a moderate water solubility of 730 mg/L at 25°C (based on CMC) and log Kow of 2.52 value, which was determined using estimation method based on solubility ratios.

Based on the R7.C indicative criteria and considering that the test substance is highly ionic, the test substance is not expected to be readily absorbed from the gastrointestinal tract.

Based on experimental data on read across substances:

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

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

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

Assessment from biocides assessment report available on read across substances:

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

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

Dermal absorption

Based on physicochemical properties:

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

The test substance is a solid paste, with an MW exceeding 100 g/mol, moderate water solubility and an estimated log Kow between 2 and 3. This together with the fact that the test substance is highly ionic suggests that the test substance is likely to have a low penetration potential through the skin.

Based on QSAR prediction:

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

In the absence of experimental data, Jmax can be calculated by multiplying the estimated water solubility with the Kp values from DERMWIN v2.01 application of EPI Suite v4.1. The calculated Jmax values for the different carbon chains of the UVCB substance was determined to be range between 0.018 to 5.89 μg/cm2/h, leading to a weighted average value of 0.49 μg/cm2/h. As per Kroes et al. 20104 and Shenet al.2014, the default dermal absorption for substances with Jmax between >0.1 to ≤10 μg/cm2/h can be considered to be less than 40%. Based on this, the test substance can be predicted to have low to moderate absorption potential through the dermal route.

Based on experimental data on read across substances:

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

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

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

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

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

Assessment from biocides assessment report available on read across substances:

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

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

Inhalation absorption

Based on physicochemical properties:

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

The test substance, because of its relatively low vapour pressure of 0.0058 Pa at 25°C, will not be available as vapours for inhalation under ambient conditions. Therefore, the substance will neither be available for inhalation as vapours nor as aerosols. Further, if at all there is any inhalation exposure, considering the moderate water solubility of the substance, it is expected to be retained in the mucus to an extent and some amount may reach the lower respiratory tract. The absorption fate of the deposited material thereafter is expected to be similar to the oral route/gastrointestinal tract.

Conclusion: Based on all the available weight of evidence information, together with the highly ionic nature, the test substance can be expected to have moderate to high absorption through the inhalation route. Therefore, as a conservative approach, a default value of 100% has been considered for the risk assessment.

METABOLISM:

Based on identified literature:

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

Based on QSAR modelling:

The OECD Toolbox (v.3.4) and FAME 2 were used to predict the first metabolic reaction, since the rat liver S9 metabolism simulator performs predictions for salts, while SMARTCyp and MetaPrint2D are not powered enough for this type of substances. The second simulator of the OECD Toolbox (in vivorat metabolism simulator) was not used as it does not consistently perform predictions for salts. As per the rat liver S9 metabolism simulator, the major constituents are primarily predicted to undergo ω or ω-1 aliphatic hydroxylation reactions. Similar results were found with FAME 2 metabolism simulation tool (which currently covers only CYP metabolism). See the 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 are also indicative of the extent of distribution. 

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

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

EXCRETION:  

Based on physicochemical properties:  

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

Based on experimental data on read across substances:  

Based on the evidence from the available oral studies (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


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