Registration Dossier

Administrative data

Description of key information

An OECD 408 study performed under GLP with C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) is available with reliability rating 1. In this study a NOAEL of 24 mg/kg was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 24 July 2015 and 09 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
A correction for purity was made taking into account of 75% purity for the test item.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories UK Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 191 to 237 g, the females weighed 145 to 184 g, and were approximately six to eight weeks old.

The animals were housed in groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).

The animals were allowed free access to food and water. A pelleted diet (Rat and Mouse SQC Ground Diet No. 1, Harlan Laboratories UK Limited, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited (formerly known as Harlan Laboratories Ltd.), Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: feed
Details on route of administration:
The test item was administered continuously, for ninety consecutive days, by dietary admixture. Control animals were treated in an identical manner with basal laboratory diet.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test item was incorporated into the diet at concentrations of 150, 400 and 1500 ppm A.I. as follows:
A known amount of test item was mixed with a small amount of basal laboratory diet at a constant speed, setting 1 in a Robot Coupe Blixer 4 mixer. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a sixty minutes at a constant speed, setting 1 in a Hobart H800 mixer.
The stability and homogeneity of the dietary admixtures was determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results showed the dietary admixtures to be stable for 17 days at room temperature and frozen. Dietary admixtures were prepared fortnightly and stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples of dietary admixture were taken on four occasions and analyzed for concentration of Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) at Envigo Research Limited, Shardlow,UK, Analytical Services.

The results indicate that the prepared formulations were within acceptable ranges of for the purpose of this study.
Duration of treatment / exposure:
Ninety consecutve days
Frequency of treatment:
Continuous
Dose / conc.:
150 ppm
Remarks:
equivalent to 9 mg/kg bw/day for males and 11 mg/kg bw/day for females
Dose / conc.:
400 ppm
Remarks:
equivalent to 24 mg/kg bw/day for males and 27.8 mg/kg bw/day for females
Dose / conc.:
1 500 ppm
Remarks:
equivalent to 92 mg/kg bw/day for males and 112.2 mg/kg bw/day for females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
The test item was administered by continuous dietary admixture to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 150, 400 and 1500 ppm (Active Ingredient (A.I.)) (equivalent to a mean achieved dosage of 9.0, 24.0 and 92.0 mg/kg bw/day and 11.0, 27.8 and 112.2 mg/kg bw/day for males and females respectively). A control group of ten males and ten females were treated with basal laboratory diet.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during Week 12 of the study.

All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.
Positive control:
N/A
Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change daily from the start of treatment; see deviations from Study Plan. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior the start of treatment) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study; see deviations from Study Plan.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 12, together with an assessment of sensory reactivity to different stimuli. Observations were carried out at a similar time on each occasion wherever possible.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion, under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and pupillary reflex. Following pupil dilation with 0.5 % Tropicamide solution (Mydriacyl® 0.5 %, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

Estrous Cycle Assessment
Vaginal smears were taken daily for 21 days, on all test and control group females during the final three weeks of the study. The stage of estrus was recorded for each day.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Terminal Investigations
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Sperm Analysis
For all test and control males, the following procedures were performed at necropsy.
i) The left testis and epididymis was removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissue was thawed and homogenized in a suitable saline/detergent mixture. Samples of the homogenate were examined microscopically to determine the number of homogenization resistant spermatids present; see
deviations from Study Plan.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyser to determine the numbers of motile, progressively motile and non-motile sperm.
iv) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then be frozen at approximately -20ºC. The tissue was later thawed and homogenized in an appropriate saline/detergent to determine the numbers of homogenization resistant spermatids.
v) Morphological assessment was performed on a sample of a minimum of 200 sperm to determine the number with apparent structural anomalies.
For ii), iv) and v) above, samples from Groups 1 and 4 only were evaluated. As there were no treatment-related findings, examinations was not extended to the remaining dose groups.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Ovaries
Brain Spleen
Epididymis (Right) Testis (Right)
Heart Thymus
Kidneys Uterus
Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymis ♦ Skin
Esophagus Spinal cord (cervical, mid-thoracic and lumbar)
Eyes*
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes (Right)♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi) # Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary glands Uterus (with cervix)
Muscle (skeletal)• Vagina
• = These tissues were retained only and not processed
♦ = Preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours
later
* = Eyes fixed in Davidson’s fluid
# = Lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before
immersion in fixative

All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues from all control and 1500 ppm A.I. dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1500 ppm A.I. males were stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the stomach, kidneys, caecum and adrenal glands (both sexes) and the liver and mesenteric lymph nodes (males only) from animals in the low and intermediate dose groups.
Sacrifice and pathology:
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Pathology
Microscopic examination was conducted by the Study Pathologist (Wendy Henderson). A peer review of the findings observed was conducted by V Mowat (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) at the histopathology peer review test site.
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.

See 'Any other information' below for more details.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 1500 ppm A.I., two males and all females showed clinical signs of fur staining towards the end of the treatment period. Such observations indicate lack of proper grooming and are considered to represent the poor clinical conditions of animals.

At 1500 ppm A.I., one male and one female showed signs of increased salivation between Days 74 to 91 and Days 73 to 77, respectively. Another male from this dose group showed signs of diarrhoea between Days 82 and 83. In isolation, these signs are considered unlikely to be related to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 1500 ppm A.I. showed a marked reduction in overall body weight gains, with a difference of approximately 45% when compared to controls. When compared with the respective controls, animals generally showed lower body weight gains throughout the treatment period achieving statistical significance in some instances. Taking into consideration the magnitude of the overall effect and the lack of recovery, this observation was considered to be of an adverse nature.

At 400 or 150 ppm A.I., animals of either sex occasionally showed reduced body weight gains with males achieving statistically significance in some instances. This resulted in marginal reductions in overall weight gains for males receiving 150 ppm A.I. and females treated with 400 or 150 ppm A.I. There was however, no clear dose relationship and these findings were deemed unlikely to be of any toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1500 ppm A.I., males generally showed lower food consumption throughout the dosing period when compared to controls. In comparison, food consumption for the corresponding females was generally similar to controls. Food conversion efficiency for these animals was generally lower than controls, reflecting intergroup differences in body weight gains and/or food intake.

At 400 or 150 ppm A.I., food consumption and food conversion efficiency for animals of either sex was generally similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of the water residues did not indicate any effect of treatment on water intake throughout the study for animals of either sex at dosages of 150, 400 or 1500 ppm A.I.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment related ocular effects for either sex at dosages of 1500, 400 or 150 ppm A.I.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females treated with 1500 ppm A.I. showed a statistically significant increase in total leukocyte counts which were mainly due to increases in neutrophil or lymphocyte counts although the latter did not achieve statistical significance in either sex. The majority of individual leukocyte or neutrophil counts together with some lymphocyte values in animals of both sexes exceeded the background control ranges. Taking into consideration the histopathological findings in the stomach and caecum, these findings may be of possible toxicological significance. Group mean monocyte counts in females treated with 1500 or 400 ppm A.I. were also statistically significantly lower than controls; this was considered to be incidental as all individual values for the test item treated females were within the
background control data ranges whilst some control females showed values that exceeded these ranges.

At 1500 ppm A.I., females showed a statistically significant reduction in hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration when compared to controls. Group mean hemoglobin level and mean corpuscular hemoglobin concentration in females from the 400 ppm A.I. dose group were also statistically significantly lower than controls with the latter also achieving statistically significant reduction in females treated with 1500 ppm A.I. The majority of individual values were within the control background data ranges and with the exception of mean corpuscular hemaglobin concentration, the remaining observations were not dose-dependant. The corresponding parameters in males were comparable to control and these observations in females were considered unlikely to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1500 ppm A.I., males showed an increase in levels of aspartate aminotransferase and alanine aminotransferase when compared to controls. The majority of the individual values were above the background control ranges. This finding correlated with the histopathological changes observed in the liver, from these males and was considered to be adaptive in nature.

Males treated with 1500 ppm A.I. showed a statistically significant increase in urea. There was no effect on the associated creatinine concentration in these males whilst the corresponding values in females from this dose group were similar to controls. Although all individual urea values in test item treated males were within the background control ranges taken together with the degeneration changes in kidneys this finding may be possible toxicological significance.

Animals of either sex treated with 1500 ppm A.I. and males receiving 400 and 150 ppm A.I. showed statistically significant reductions in albumin levels in relation to the respective controls. No dose-dependence was evident in males. Males from the 1500 ppm A.I. dose group also showed a statistically significant reduction in albumin/globulin ratio. All individual values were within the background control ranges and taking into consideration the microscopic changes in the liver from the high dose males these findings may represent minor perturbations in metabolic pathways and were considered to be of an adaptive nature.

At all dosages males showed statistically significant reduction in phosphorus levels relative to controls with males from the 1500 or 400 ppm A.I. also showing statistically significant reduction in chloride levels. All individual values were within the background control ranges whilst the corresponding values in the females were similar to controls. Whilst these observations may be limited to adaptive changes in adrenals seen in the high dose animals they were deemed unlikely to be of any toxicological significance.

Any other intergroup differences attaining statistical significance were confined to males from 150 or 400 ppm A.I. dose groups and were deemed likely to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in behavioural parameters measured.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males from all treatment groups showed a statistically significant reduction in liver weight both absolute and relative to terminal body weight. There was no dose-relationship for the body weight-related liver weights and the majority of the individual values were within the background control ranges. Macroscopic examination of the liver revealed periportal atrophy for males treated with 1500 ppm A.I. and this observation was deemed to be of an adaptive nature.

Females treated with 1500 or 400 ppm A.I. showed a statistically significant increase in spleen weights both absolute and relative to terminal body weight. All individual values were within the background control ranges. There was no histopathological findings to correlate this, therefore this was considered of no toxicological importance.

At 400 ppm A.I. males showed a statistically significant increase in kidney weights both absolute and relative to terminal body weight. The majority of individual values were within the background control ranges. As there were no histopathological findings at this level, this was considered incidental.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All males and eight females treated with 1500 ppm A.I. had a raised non glandular region of the stomach at necropsy. These findings correlated with histopathological alternations noted for the high dose animals of both sexes.

A number of animals had reddened lungs at necropsy. Such findings are common in this type of study and in the absence of a dose-relationship or histopathological correlates these findings were considered un-related to treatment with the test item.

One female treated with 150 ppm A.I. showed an enlarged right kidney and a fibrous mass on the left kidney. In isolation, this finding was deemed incidental.

No toxicologically significant effects were detected in animals of either sex treated with 400 or 150 ppm A.I.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Sensory Reactivity Assessments There were no treatment-related changes in sensory reactivity.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Adrenal Gland
Hypertrophy of the zona glomerulosa was present in 5/10 males and females treated with 1500 ppm A.I. This finding was not present in animals treated with 400 or 150 ppm A.I.

Caecum
Hyperplasia of the mucosa of the caecum was present in all males and 7/10 females treated
with 1500 ppm A.I. Inflammation was present in all males and 6/10 females. Hyperplasia of
the caecum was present in one female treated with 400 and 150 ppm A.I.

Kidneys
At 1500 ppm A.I. various changes were apparent in most males and all females. These
included tubular dilation, especially in the medullary tubules, increased basophilic tubules
and brown pigment deposition. No such changes were apparent in animals of either sex
treated with 400 or 150 ppm A.I.

Liver
Periportal atrophy (smaller, darker staining cells) was present in 5/10 males treated with 1500 ppm A.I. This was not present in females at 1500 ppm A.I. or males treated with 400 or 150 ppm A.I.

Mesenteric Lymph node
Erythrocytosis was present in 6/10 males treated with 1500 ppm A.I. compared to 1/10 controls. This was not present in males treated with 400 or 150 ppm A.I.

Stomach
At 1500 ppm A.I. the changes present in the stomach of all males and 9/10 females. The changes included ulceration, erosion and various degrees of hyperplasia, all affecting the nonglandular region. The stomachs of animals of either sex treated with 400 or 150 ppm A.I. were within expected limits.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Functional Performance Tests
There were no toxicologically significant changes in functional performance.

At all dose levels, males showed statistically significant increase in forelimb grip strength during Week 12 of the treatment period. There was, however, no dose relationship and all individual values were within the historical control data ranges. Additionally, forelimb grip strength for these males in 2/3 tests was similar to controls and this observation was therefore considered likely to be incidental.

Sperm Analysis
At necropsy, sperm analysis did not indicate any appreciable differences in group mean sperm concentration and motility at any dose level. An evaluation of homogenization resistant spermatid counts and morphology in males from the control and 1500 ppm A.I. dose groups also did not reveal any treatment-related differences.

Estrous Cycle Assessment
There was no effect of treatment at any dose level on estrous cycling activity in females as assessed over the last three weeks of dosing.
Details on results:
DISCUSSION
The continuous oral (dietary) administration of Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) to rats, at dietary concentrations of 150, 400 and 1500 ppm A.I., for ninety consecutive days resulted in marked reduction in body weight gains for animals of either sex treated with 1500 ppm A.I. and lower food consumptions for males at this level. Taking into account the overall magnitude of the reduction in overall body weight gain and lack of recovery these findings were considered to be adverse.

Males and females treated with 400 or 150 ppm A.I. showed occasional fluctuations in body weight gains, however, there was no dose relationship and these findings were considered to be of no toxicological significance. Food consumption and food conversion efficiency for
these animals were similar to controls.

The changes in the stomach were indicative of an irritant effect on the non-glandular area of the stomach which is specific to rodents. This structure is not present in humans and so this observation was considered to be of no relevance for extrapolation to human exposure;
irritation in the stomach may reflect an inherent characteristic of the test item which is not indicative of systemic toxicity.

The caecal changes for the 1500 ppm A.I. animals of either sex may be due to a direct irritant effect or related to an upset in the flora or contents of the intestine. The inflammation observed in the caecum would correlate with the elevated levels of leucocytes, neutrophils and lymphocytes. These changes were considered to be of toxicological significance. The changes in one female from each of 400 and 150 ppm A.I. level showed no dose related trend and are considered to be incidental.

The changes in the kidney at 1500 ppm A.I. are degenerate in nature and likely to be directly related to the administration of the test item or related to the effect of excretion of the test item or its metabolites.

Erythrocytosis in the mesenteric lymph node was of unknown etiology but could possibly be related to the changes seen in the gastrointestinal tract; especially the caecum. The toxicological significance of this finding remaning inconclusive.

The adrenal gland zona glomerulosa hypertrophy was considered to be of an adaptive change and may be linked to changes in hydration or electrolyte balance. The microscopic changes in livers of males given 1500 ppm A.I. were considered to be an adaptive response to
metabolism of the test item and not necessarily indicative of systemic toxicity. These changes correlated with increased aspartate and alanine aminotransferase activities for these males and reduced liver weights. This can be associated with the periportal atrophy seen at
the histopathological examination seen in half of the 1500 ppm A.I. males only.

Overall, it is considered that a No Observed Adverse Effect Level (NOAEL) for systemic toxicity for animals of either sex would be 400 ppm A.I. and equivalent to a mean achieved dosage of 24.0 mg/kg bw/day for males and 27.8 mg/kg bw/day for females within the confines of this study.
Key result
Dose descriptor:
NOAEL
Effect level:
400 ppm
Based on:
test mat.
Remarks:
equivalent to 24 mg/kg bw/day for males and 27.8 mg/kg bw/day for females
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm
System:
urinary
Organ:
kidney
liver
mesenteric lymph node
stomach
other: caecum
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no

Achieved Intakes

Group mean achieved dosages of Quaternary ammonium compounds, C12-18- alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) in mg/kg bw/day during the study are given in Table 1 and are calculated using nominal concentrations for dietary formulations.

At 1500 ppm A.I., the mean achieved intake for males was 92.0 mg/kg bw/day and for females it was 112.2 mg/kg bw/day. Overall, animals of either sex achieved approximately 10.2 fold interval between the high dietary level and the low dietary level.

At 400 ppm A.I., the mean achieved intake for males was 24.0 mg/kg bw/day and for females it was 27.8 mg/kg bw/day. Overall, males achieved approximately 2.7 fold interval between the intermediate dietary level and the low dietary level whilst for females the interval was approximately 2.5 fold.

At 150 ppm A.I., the mean achieved intake for males was 9.0 mg/kg bw/day and for females it was 11.0 mg/kg bw/day.

Conclusions:
The continuous oral (dietary) administration of Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) to rats, at a dietary concentration of 1500 ppm A.I. for ninety consecutive days, at 1500 ppm A.I. resulted in animals of either sex showing an adverse reduction in body weight gains and reduced food consumptions in the males. The histopathological findings observed at this dose level in animals of both sexes included adverse changes in the kidneys, stomach and caecum with some males also showing erythrocytosis in mesenteric lymph node, a change that may be associated with changes with caecal changes. In addition, changes to the adrenal glands and liver were deemed likely to be adaptive in nature.

There were no toxicologically significant effects for animals of either sex at dose levels of 400 or 150 ppm A.I. and therefore a No Observed Adverse Effect Levels (NOAEL) for systemic toxicity for animals of either sex was considered to be 400 ppm A.I., equivalent to a mean achieved dosage of 24.0 mg/kg bw/day for males and 27.8 mg/kg bw/day for females within the confines of this study.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the requirements of the OECD guidelines for Testing of Chemicals No. 408 “Subchronic Oral Toxicity - Rodent: 90 Day Study” (adopted 21 September 1998). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by continuous dietary admixture to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 150, 400 and 1500 ppm (Active Ingredient (A.I.)) (equivalent to a mean achieved dosage of 9.0, 24.0 and 92.0 mg/kg bw/day and 11.0, 27.8 and 112.2 mg/kg bw/day for males and females respectively). A control group of ten males and ten females were treated with basal laboratory diet.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during Week 12 of the study.

All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

At 1500 ppm A.I., two males and all females showed clinical signs of fur staining towards the end of the treatment period. Such observations indicate lack of proper grooming and are considered to represent the poor clinical condition of the animals.

There were no other signs of toxicological significance.

Behavioral Assessment

There were no treatment-related changes in behavioral parameters measured.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Body weight gain was noticeably lower throughout the study for animals of either sex treated with 1500 ppm A.I. when compared to controls. Overall body weight gain was 45% lower for both males and females at this level. These findings were considered to be of an adverse nature.

There were no adverse effects of treatment with the test item at dose levels of 400 or 150 ppm A.I. in animals of either sex.

Food Consumption

Males treated with 1500 ppm A.I., showed a reduced food intake throughout the study. There were no effects detected in females treated at 1500 ppm A.I. or animals of either sex treated with 400 or 150 ppm A.I.

Animals of either sex treated with 1500 ppm A.I. showed reduced food conversion efficiency throughout the study when compared to controls. The corresponding values for animals of either sex treated with 400 or 150 ppm A.I. were generally similar to controls.

Water Consumption

There were no treatment-related effects detected in water consumption.

Ophthalmoscopy

There were no treatment-related ocular effects detected.

Estrous Cycle Assessment

There was no effect of treatment with the test item on estrous cycling activity assessed over the last three weeks of dosing in females.

Hematology

Males and females treated with 1500 ppm A.I. showed a statistically significant increase in total leukocyte count and neutrophils when compared to controls. Group mean lymphocyte values in these animals were also higher than controls but without attaining statistical significance. The majority of individual values exceeded the background control ranges, and these observations were deemed to be of toxicological significance.

There were no other toxicologically significant effects on the hematological parameters measured for animals of either sex treated with 400 or 150 ppm A.I.

Blood Chemistry

Males treated with 1500 ppm A.I. showed statistically significant increase in aspartate aminotransferase and alanine aminotransferase activity. The majority of the individual values were above the background control ranges, and these findings were considered to be related to adaptive microscopic changes in the liver from these males.

Males treated with 1500 ppm A.I. showed a statistically significant increase in urea. There was no effect on the associated creatinine concentration in these males whilst the corresponding values in females from this dose group were similar to controls. Although all individual urea values in test item treated males were within the background control ranges taken together with the degeneration changes in kidneys this finding may possibly be toxicologically significant.

There were no other toxicologically significant effects on blood chemistry parameters measured on animals of either sex treated with 400 or 150 ppm A.I.

Necropsy

All males and eight females treated with 1500 ppm A.I. had a raised non glandular region of the stomach at necropsy, this correlated with microscopic changes in the non-glandular region of the stomach at this dose level.

No other treatment related findings were observed at any dose level.

Organ Weights

There were no toxicologically significant effects detected in the organ weights measured.

Sperm Analysis

Analyses of sperm concentration, motility, morphology and homogenisation resistant spermatid counts did not identify any treatment-related differences.

Histopathology

The following treatment related microscopic abnormalities were detected:

Adrenal Gland: Hypertrophy of the zona glomerulosa was present in 5/10 males and females treated with 1500 ppm A.I. This finding was considered to be of adaptive in nature.

Stomach: Changes were present in the stomach of all males and 9/10 females treated with 1500 ppm A.I. The changes included ulceration, erosion and various degrees of hyperplasia, all affecting the non-glandular region. This finding was considered to be adverse but not linked to man.

Caecum: Hyperplasia of the mucosa of the caecum was present in all males and 7/10 females treated with 1500 ppm A.I., also inflammation was present in all males and 6/10 females. These findings may be due to direct irritation or upset in the flora or contents in the intestines.

Kidneys: Various changes were apparent in most males and all females treated with 1500 ppm A.I. These included tubular dilation, especially in the medullary tubules, increased basophilic tubules and brown pigment deposition. This finding was considered to be degenerative.

Liver: Periportal basophilia (homogenous, darker staining cells) was present in 5/10 males treated with 1500 ppm A.I. This finding was considered to be of adaptive in nature.

Mesenteric Lymph node: Erythrocytosis was present in 6/10 males treated with 1500 ppm A.I. compared to 1/10 controls. This finding was considered to be an unknown etiology.

Conclusion

The continuous oral (dietary) administration of Quaternary ammonium compounds, C12-18- alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) to rats, at a dietary concentration of 1500 ppm A.I. for ninety consecutive days, at 1500 ppm A.I. resulted in animals of either sex showing an adverse reduction in body weight gains and reduced food consumptions in the males. The histopathological findings observed at this dose level in animals of both sexes included adverse changes in the kidneys, stomach and caecum with some males also showing erythrocytosis in mesenteric lymph node, a change that may be associated with changes with caecal changes. In addition, changes to the adrenal glands and liver were deemed likely to be adaptive in nature.

There were no toxicologically significant effects for animals of either sex at dose levels of 400 or 150 ppm A.I. and therefore a No Observed Adverse Effect Levels (NOAEL) for systemic toxicity for animals of either sex was considered to be 400 ppm A.I., equivalent to a mean achieved dosage of 24.0 mg/kg bw/day for males and 27.8 mg/kg bw/day for females within the confines of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
24 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is performed according to OECD 408 guideline and under GLP and has reliability rating 1. It is sufficient to cover the information requirements in Annex IX.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 90 -day oral toxicity feeding study according to OECD 408 has been performed on C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2). The dose levels tested were 150 ppm (9 mg/kg bw in males, 11 mg/kg bw in females), 400 ppm (24 mg/kg bw in males and 27.8 mg/kg bw in females) and 1500ppm (92 mg/kg bw in males and 112.2 mg/kg bw in females). This study was performed according to the current OECD 408 protocol, with full GLP compliance and a well-defined test substance.

The continuous oral (dietary) administration of Quaternary ammonium compounds, C12-18 -alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) to rats, at a dietary concentration of 1500 ppm A.I. for ninety consecutive days, at 1500 ppm A.I. resulted in animals of either sex showing an adverse reduction in body weight gains and reduced food consumptions in the males. The histopathological findings observed at this dose level in animals of both sexes included adverse changes in the kidneys, stomach and caecum with some males also showing erythrocytosis in mesenteric lymph node, a change that may be associated with changes with caecal changes. In addition, changes to the adrenal glands and liver were deemed likely to be adaptive in nature.

There were no toxicologically significant effects for animals of either sex at dose levels of 400 or 150 ppm A.I. and therefore a No Observed Adverse Effect Levels (NOAEL) for systemic toxicity for animals of either sex was considered to be 400 ppm A.I., equivalent to a mean achieved dosage of 24.0 mg/kg bw/day for males and 27.8 mg/kg bw/day for females within the confines of this study.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The most relevant available repeated dose oral toxicity study on C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) is performed according to OECD 408 guideline under GLP conditions and has reliability rating 1.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

There is no repeat dose inhalation study on C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) however the substance has low wapour pressure < 0.00073 Pa at 20 °C. Significant exposure to vapours would not be expected at ambient temperatures so it is considered to not be scientifically valid to conduct conduct a repeat dose inhalation study. Also the corrosive nature of the substance would make it difficult to perform such a test for animal welfare reasons. The guidance of REACH allows the inhalation long term DNEL to be calculated based on the oral repeat dose NOAEL.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

There is no repeat dose inhalation study on C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) however the substance has low wapour pressure < 0.00073 Pa at 20 °C. Significant exposure to vapours would not be expected at ambient temperatures so it is considered to not be scientifically valid to conduct conduct a repeat dose inhalation study. Also the corrosive nature of the substance would make it difficult to perform such a test for animal welfare reasons. The low possibility of inhalation makes such a test scientifically unjustified.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

There is no repeat dose dermal study C12-18-alkylbis(hydroxyethyl)methyl, chloride, CAS no 71808-53-2, and due to the corrosive properties of the substance it is not possible to conduct repeat dose dermal toxicity studies due to animal welfare considerations. It is also considered very unlikely that dermal absorption would exceed oral absorption, so it would be expected than the oral NOAEL would be lower than a corresponding value from a dermal study. Results from the available repeat dose oral study can be used in the setting of DNELs in accordance with the REACH guidelines. As appropriate DNEL values can be calculated using the oral dosing study data, it is not justified on animal welfare grounds to perform a repeat dose dermal toxicity study.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

There is no repeat dose dermal study on C12-18-alkylbis(hydroxyethyl)methyl, chloride, CAS no 71808-53-2, and due to the corrosive properties of the substance risk management measures such as wearing appropriate gloves and protective clothing will prevent any significant dermal contact. Local effects on the skin would be expected to be limited to local irritation /corrosion, which would be dependent on the local concentration rather than the dose or duration of exposure. This is a medium hazard and does not justify the requirement for an additional dermal animal study to establish a local NOAEL for dermal exposure.

Justification for classification or non-classification

Based on the lack of any findings relevant for human health from the available sub-chronic oral toxicity study, there is no requirement for a classification for any STOT category under the EU CLP (GHS) criteria.