Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 02 November 2016. Experimental Completion Date: 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Identification: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1, N1-dimethyl-1, 3-propanediamine (ACAR 16001)
Physical State/Appearance: Dark Amber coloured liquid
Purity: 59.7% (sample supplied as solution containing 40% propylene glycol)
Batch Number: 2629-70-10
Label: ANSC (RDI): ACAR 16001 2629-70-10
Date Received: 24 May 2016
Storage Conditions: Room temperature, in the dark
Expiry Date: 18 January 2019
A correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 285 to 365g and were approximately eleven weeks old. The females weighed 189 to 242g, and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. 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) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. 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, 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; there were no deviations from these targets.
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.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxi
city of the test item to man.
Vehicle:
polyethylene glycol
Details on oral exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Due to the method of analysis used on the study, stability could not be determined as this method was non-stability indicating. Therefore, formulations were prepared daily and administered within two hours of preparation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement, also see deviations from study plan.
Samples of test item formulations were taken on seven occasions and analyzed for concentration of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within ± 10% of the nominal concentration, with the exception of analysis 2. The results of analysis 2 revealed the concentrations of the intermediate and high dose levels were within 19% of the nominal concentration. These results do not impact on the study as the majority of the formulations were within 10% of the nominal concentration and this was an isolated occurance.
Duration of treatment / exposure:
approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females) at dose levels of 40, 100 and 250 mg/kg bw/day of Active Ingredient (A.I.) (incorporating a correction factor for 59.7% purity). A control group was dosed with vehicle alone (Polyethylene glycol 400).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
67 mg/kg bw/day (nominal)
Dose / conc.:
167.5 mg/kg bw/day (nominal)
Dose / conc.:
418.8 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
active ingredient
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
active ingredient
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
active ingredient
No. of animals per sex per dose:
12 males and females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work including a 14 day dose range-finding toxicity study in the rat (Envigo Study Number: TY70LJ). During this range finder study, one male was killed in extremis on Day 3 of treatment due to excessive clinical signs and the remaining animals of either sex treated with 500 mg/kg bw/day A.I. were prematurely terminated on Day 4 due to marked body weight loss. At 250 mg/kg bw/day A.I., males showed a slightly lower overall body weight gain and marginally lower food consumption. No clinical signs of toxicity were evident at 250 mg/kg bw/day A.I. nor any macroscopic findings.

Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.
iii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
vi. On completion of the pairing phase, five selected males per dose group were evaluated for functional/sensory responses to various stimuli during Week 6.
vii. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
viii. At Day 12 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 or 45.
x. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 13 post partum. At Day 13 post partum, all surviving offspring were killed and examined macroscopically. All females were killed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also killed and examined macroscopically around the same time as littering females.
xi. Where possible, blood samples were taken from two offspring on Day 4 post partum and one male and one female offspring on Day 13 post partum. In addition, blood samples were taken from all adult males and females at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase, females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Estrous Cycle Assessment
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

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. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute 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 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. The animal was pulled by the base of the tail until its grip was broken. 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).
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
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)
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++)

Thyroid Hormone Analysis
Where possible, blood samples were taken, allowed to clot, centrifuged, and the serum from each blood sample stored frozen at lower than -60 °C for possible evaluation of thyroid hormones. Blood samples were also taken to produce plasma, collected in K2EDTA, centrifuged, and the plasma from each blood sample stored frozen below -60 ºC for possible evaluation of further thyroid hormones. Samples were taken as follows:
Two offspring from each litter at Day 4 post partum (serum only).
One male and one female offspring from each litter at Day 13 post partum.
All adult males at termination.
All adult females at termination.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (H Bose).
Sacrifice and pathology:
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy were killed around the same time as littering females.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. All offspring were subjected to an external examination. Any decedent offspring or offspring showing macroscopic abnormalities were also examined internally.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues marked with a * were weighed from all remaining animals:
Adrenals
Pituitary (weighed post-fixation)*
Brain
Prostate and Seminal Vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)*
Ovaries*
Uterus (weighed with Cervix and Oviducts)*

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals
Mammary gland
Aorta (thoracic)
Muscle (skeletal)
Bone & bone marrow (femur including stifle joint)
Ovaries
Bone & bone marrow (sternum)
Pancreas
Brain (including cerebrum, cerebellum and pons)
Pituitary
Caecum
Prostate
Coagulating gland●
Rectum
Colon
Salivary glands (submaxillary)
Cowpers glands●
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides ♦
Skin
Esophagus
Spinal cord (cervical, mid thoracic
Eyes *
and lumbar)
Glans penis●
Spleen
Gross lesions
Stomach
Heart
Testes ♦
Ileum (including peyer’s patches)
Thyroid/Parathyroid
Jejunum
Trachea
Kidneys
Thymus
LABC (levator ani-bulbocavernous) muscle●
Urinary bladder
Liver
Uterus & Cervix
Lungs (with bronchi)#
Vagina
Lymph nodes (mandibular and mesenteric)
Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues from five selected control and 250 mg/kg bw/day 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. The tissues shown in bold from the remaining control and 250 mg/kg bw/day A.I. animals, any animals which failed to mate or animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 250 mg/kg bw/day A.I. males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
● = Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by Vasanthi Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post Implantation Loss, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Episodes of increased salivation and noisy respiration were observed between Study Days 1 and 43 among individuals of either sex treated at 100 and 250 mg/kg bw/day A.I. No such effects were detected for the 40 mg/kg bw/day A.I. animals of either sex.
In the absence of supporting evidence of systemic toxicity, observations of this nature are considered to be associated with gavage administration of slightly irritant or unpalatable test formulations and in isolation are therefore considered not to be of toxicological importance.
Incidental changes included pilo-erection and hunched posture discovered in one 250 mg/kg bw/day A.I. female between Days 41 and 47. An incident of diarrhoea recorded on Days 41/42 for one 250 mg/kg bw/day A.I. female and staining around the snout of one 100 mg/kg bw/day A.I. female on Day 4. At 40 mg/kg bw/day A.I. noisy respiration was observed in one male on Day 10 only with staining around the eyes also noted in this male between Days 38 and 39. The sporadic and transient nature of these intergroup findings would suggest they are of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg bw/day A.I., males showed a reduction in body weight gain during Weeks 1 and 2 of treatment. Improvement was evident during Weeks 3 and 4, however, a statistically significant reduction (p<0.01) was evident in these males during Week 5. Overall body weight gain was also 23% lower than controls.
No such effects were evident in males treated with 100 or 40 mg/kg bw/day A.I.
During the pre-pairing phase, females treated with 250 mg/kg bw/day A.I. showed a reduction in body weight gain during the first week (although statistical significance was not achieved). Improvement was noted during the second week as body weight gain was similar to controls. There was no adverse effect on body weight development for any treated female during gestation or lactation.
Females treated with 40 mg/kg bw/day A.I. showed a slight reduction in body weight gain during the first week of treatment. A true dose-related response was not evident and body weight gain for the remainder of the treatment period was comparable to controls. Therefore, this was considered to be the result of normal biological variation. There was a statistically significant increase (p<0.05) in cumulative body weight gain during Days 0 and 14 of gestation for females receiving 100 mg/kg bw/day A.I. An increase in body weight gain is considered not to reflect an adverse effect of treatment, therefore the intergroup difference was considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 250 mg/kg bw/day A.I. showed a slight reduction in food consumption throughout the treatment period. Overall food consumption for these males was 7% lower than controls. No such effects were detected in females treated with 250 mg/kg bw/day A.I. or in animals of either sex treated with 100 or 40 mg/kg bw/day A.I.
Minor fluctuations in food conversion efficiency were evident in animals of either sex treated with 250 mg/kg bw/day A.I., which generally followed the reductions in body weight gains seen in these animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in the hematological parameters examined.
Males from all treatment groups showed a statistically significant increase (p<0.05 to p<0.01) in reticulocyte count in a dose related manner. However, while the aetiology is uncertain, in the absence of any supporting evidence of treatment-related histopathological findings the intergroup differences were considered not to be of toxicological importance.
A statistically significant increase in reticulocyte count (p<0.01) was noted for females treated at 250 mg/kg bw/day A.I. However, as all individual values were within the anticipated historical control range and in the absence of any supporting evidence of an effect on hemopoiesis, this isolated change was considered not to be of toxicological significance.
At 250 or 100 mg/kg bw/day A.I. males showed a statistically significant decrease (p<0.05) in prothrombin time. However, as individual values were within the background control ranges and there were no concomitant changes in supporting platelet or activated partial thromboplastin times, this isolated finding was therefore considered to be of no toxicological importance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.
All male treated groups showed a statistically significant decrease (p>0.05 or p>0.01) in potassium and phosphorous levels. The individual values were within the background control ranges for these parameters. Males treated with 100 or 250 mg/kg bw/day A.I. showed a stastistically significant increase (p>0.01 and p>0.05, respectively) in albumin and chloride levels when compared to controls. All the individual values for albumin were within the background control ranges. However, for chloride concentration, one and three of the individual values for 100 and 250 mg/kg bw/day A.I. males were above the background control ranges. Also at 250 mg/kg bw/day A.I. males showed an increase (p>0.05) in creatinine levels in relation to controls. In the absence of any supporting histopathological findings these intergroup differences were considered not to be of toxicological importance.
Females treated with 250 mg/kg bw/day A.I. showed a statistically significant increase in chloride concentration (p<0.05) and a statistically significant reduction in bilirubin (p<0.05). The majority of the individual values for chloride were outside the background control ranges, whereas all individual values for bilirubin were within the background ranges. There were no histopathological correlates and therefore these intergroup differences were considered not to be of toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at 40, 100 or 250 mg/kg bw/day A.I.
One male treated with 250 mg/kg bw/day A.I. showed noisy respiration during the final assessment on Day 40. This observation can be associated with the clinical observations and considered to be incidental.
Functional Performance Tests
There were no treatment related changes in functional performance at 40, 100 or 250 mg/kg bw/day A.I.
Statistical analysis of the data did not reveal any significant intergroup differences.
Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 40, 100 or 250 mg/kg bw/day A.I.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured in animals of either sex treated with 40, 100 and 250 mg/kg bw/day A.I.
Males treated with 250 mg/kg bw/day A.I. showed a statistically significant decrease (p<0.05) in prostate weight. All individual values are within the background control ranges. There was also a statistically significant increase (p<0.05) in thyroid weight, both absolute and relative to terminal body weight with the majority of individual values within the background control ranges. There were no histopathological correlates, therefore these intergroup differences were considered to be of no toxicological importance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in animals of either sex treated with 40, 100 and 250 mg/kg bw/day A.I.
One control male had increased pelvic space in the right kidney. In the absence of treatment, this was considered to be incidental.
One male treated with 40 mg/kg bw/day A.I. had increased pelvic space in the left kidney and a small right testis. The finding in the testis correlated to the microscopic finding of tubular degeneration in both testes. In the absence of similar effects detected at 250 mg/kg bw/day A.I. these macroscopic findings were considered to be incidental and of no toxicological significance.
Two females treated with 250 mg/kg bw/day A.I. had an enlarged spleen. One of these females also had increased pelvic space in both kidneys. A further female from this treatment group had increased pelvic space in the right kidney and a small and pale left kidney. In the absence of any treatment-related histopathological changes evident at 250 mg/kg bw/day A.I., these intergroup differences were considered not to be of toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item-related microscopic findings in the testes, including the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle). No treatment related effects were seen following the evaluation of the uterus or evaluation of ovarian follicles and corpora lutea.
Other effects:
no effects observed
Description (incidence and severity):
Estrous Cycle
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with the majority of females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating
No treatment-related effects were detected in mating performance.
One mating pair (Male 27 and Female 39) failed to show any positive evidence of mating. Microscopic examination revealed that the male had tubular degeneration in the testes and this is likely to have caused an issue with this pairing.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female (No.62 (male partner no. 50)) was found to be non-pregnant following positive evidence of mating. Histopathological examinations of the female and the respective male partner did not reveal any significant microscopic changes which could account for the lack of pregnancy. In the absence of a similar effect at 250 mg/kg bw/day A.I. the intergroup difference was considered incidental and unrelated to treatment.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was comparable to controls and gestation lengths were all between 22 and 23 days.

Thyroid Hormone Analysis
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Effect levels

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
active ingredient
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food efficiency
water consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001) to rats by gavage, at dose levels of 40, 100 and 250 mg/kg bw/day A.I., resulted in males treated with 250 mg/kg bw/day A.I. showing a lower overall body weight gain and food consumption in relation to controls (in relation to controls, body weights were 4% lower compared to controls) and the corresponding females during the first week of treatment also showed reduced body weight gains. There were no histopathological findings related to treatment at any dose level and the effect on body weight development was considered to be non-adverse. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 250 mg/kg bw/day A.I.
The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 250 mg/kg bw/day A.I.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

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 gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 40, 100 and 250 mg/kg bw/day of Active Ingredient (A.I.) (incorporating a correction factor for 59.7% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all females and all surviving offspring on Day 14 and 13 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Instances of increased salivation and/or noisy respiration were observed during the course of the study in animals of either sex treated with 250 or 100 mg/kg bw/day A.I.

No toxicologically significant effects were evident in animals treated with 40 mg/kg bw/day A.I.

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters.

Functional Performance Tests

There were no treatment related changes in functional performance.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores.

Body Weight

Males treated with 250 mg/kg bw/day A.I. showed a reduction in body weight gain during Weeks 1, 2 and 5. Consequently overall body weight gain for these males was lower than controls. Females treated with 250 mg/kg bw/day A.I. showed a reduction in body weight gain during Week 1. Recovery was evident thereafter.

No such effects were detected in animals of either sex treated with 100 or 40 mg/kg bw/day A.I.

Food Consumption

A slight reduction in food consumption was evident in males treated with 250 mg/kg bw/day A.I. throughout the treatment period. Minor fluctuations in food conversion efficiency were evident in animals of either sex treated with 250 mg/kg bw/day A.I., which generally followed the reductions in body weight gains seen in these animals. No such effect on food consumption was evident in females treated with 100 mg/kg bw/day A.I. or in animals of either sex treated with 100 or 40 mg/kg bw/day A.I. Food conversion efficiency for either sex treated with 100 or 40 mg/kg bw/day A.I. was comparable to controls.

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Reproductive Performance

Estrous Cycle

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating

There was no effect of treatment on mating performance. With the exception of one animal, all animals mated within four days of pairing.

Fertility

There were no treatment-related effects in conception rates for test item treated animals in relation to controls.

Gestation Lengths

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Pathology

Necropsy

No toxicologically significant effects were detected in animals of either sex treated with 40, 100 and 250 mg/kg bw/day A.I.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured in animals of either sex treated with 40, 100 and 250 mg/kg bw/day A.I.

Histopathology

There were no treatment-related microscopic abnormalities detected.

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Conclusion

The oral administration of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001) to rats by gavage, at dose levels of 40, 100 and 250 mg/kg bw/day A.I., resulted in males treated with 250 mg/kg bw/day A.I. showing a lower overall body weight gain and food consumption in relation to controls (in relation to controls, body weights were 4% lower compared to controls) and the corresponding females during the first week of treatment also showed reduced body weight gains. There were no histopathological findings related to treatment at any dose level and the effect on body weight development was considered to be non-adverse. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 250 mg/kg bw/day A.I.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 250 mg/kg bw/day A.I.