Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
28 November 2011 and 13 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP cobditions.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification :1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulf
onates
Chemical name :1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo- 2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulf onates
Description :amber coloured slightly viscous liquid
Purity active content 78.6%
Batch number :P1118
Date received :17 October 2011
Expiry date :01 June 2012
Storage conditions :room temperature in the dark, under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for five 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 314 to 365g, the females weighed 193 to 221g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating 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, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the 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 computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperatures and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Short term deviations from these targets were considered not to affect the purpose or integrity of the study.
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. 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
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (Harlan Laboratories Ltd., Project Number: 41101014). Results from the previous study showed the formulations to be stable for at least twenty-one days. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark under nitrogen.

Samples of each test item formulation were taken and analysed for concentration of 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulfonates at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 9% of the nominal concentration.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (Harlan Laboratories Ltd., Project Number: 41101014). Results from the previous study showed the formulations to be stable for at least twenty-one days. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark under nitrogen.

Samples of each test item formulation were taken and analysed for concentration of 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulfonates at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 9% of the nominal concentration.

The concentration of 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulfonates (PISCES 1 monomer) in the test item formulations was determined spectrophotometrically. For full details, please see attached Appendix 25.
Duration of treatment / exposure:
The oral administration of the test substance to rats for a period of up to eight weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
75 mg/kg bw/day (18.75 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day (37.5 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
350 mg/kg bw/day (87.5 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based Preliminary Fourteen Day Repeated Dose Oral (Gavage) Range-Finder in the Rat

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 bw/day 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 regular intervals.
Positive control:
Not applicable, as no positive control used.

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. 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.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour 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 Behavioural 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 each day, 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).
The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach
Normal range data for the functional performance test parameters are presented in Addendum 6.

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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
Normal range data for body weight changes in pregnant and lactating females are presented in Addendum 3.

Food Consumption
During the maturation 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 and 4 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-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Nomal range data for pregnant and lactating females are presented in Addendum 3.

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

Laaboratory Investigations
Haematological 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.

The methods used for haematological and blood chemical investigations are given in Addendum 2 and normal ranges are shown in Addendum 5.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte 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 Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Bile acids (Bile)







Sacrifice and pathology:
Pathology
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

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 and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

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 Prostate
Brain Seminal vesicles
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)
Normal ranges for these organ weights are given in Addendum 7.

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)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides• Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and
Gross lesions lumbar)
Heart Spleen
Ileum (including peyer’s patches) Stomach
Jejunum Thyroid/parathyroid
Kidneys Trachea
Liver Testes•
Lungs (with bronchi) #
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cervix
Muscle (skeletal) Vagina

All tissues were despatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford HR2 6JU) for processing. The tissues from five selected control and 350 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 350 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 350 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related changes in the stomach and spleen, examination was subsequently extended to include similarly prepared sections of the stomach and spleen from five animals per sex from the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist (Dr Yoshimasa Okazaki). A peer review of the histopathology examinations was also performed in this phase of the study.
Other examinations:
None.
Statistics:
The following statistical procedures were used:
Data for males and females prior to pairing, and functional performance test data, where appropriate, were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (p) were calculated as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Please refer to Details on Results section below
Mortality:
mortality observed, treatment-related
Description (incidence):
Please refer to Details on Results section below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to Details on Results section below
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to Details on Results section below
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Please refer to Details on Results section below
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to Details on Results section below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
One female treated with 350 mg/kg bw/day was found dead on Day 41. This death was considered to be related to the microscopic observations detected for this animal.

There were no further treatment related deaths.

One control female was found dead on Day 10. This interim death was considered to be due to an accidental oesophageal injury and respiratory tract inflammation.


Clinical Observations
A summary incidence of clinical observations is given in Table 2. Individual data are given in Appendix 1.

Episodes of increased salivation were evident in animals of either sex treated with 350 mg/kg bw/day from Day 18 (males) and Day 24 (females) onwards. Incidents of noisy respiration were also evident in four males and two females from this treatment group between Days 6 and 39. One male that showed noisy respiration also showed laboured respiration on Days 18 and 19 and a decreased respiratory rate on Day 18. The female treated with 350 mg/kg bw/day that was found dead on Day 41 only showed incidences of increased salivation prior to death.

At 150 mg/kg bw/day, increased salivation was evident in males between Days 18 and 28 and in three females on Day 28. Two males from this treatment group also showed noisy respiration on either Day 8 or 25.

Red/brown staining around the mouth was evident in one male treated with 350 mg/kg bw/day and in three males treated with 150 mg/kg bw/day on Day 15 only.

No such effects were detected in animals of either sex treated with 75 mg/kg bw/day.

The control female that was found dead on Day 10 showed noisy/laboured respiration, a decreased respiratory rate, pilo-erection and hunched posture on Day 9.


Functional Observations
Group values of behavioural assessment observations are given in Table 3 and group mean behavioural assessment scores are given in Table 4. Group mean functional test values and standard deviations are given in Table 5 (statistically significant differences are indicated). Individual values are given in Appendices 2 to 5. Group mean sensory reactivity assessment scores are given in Table 6. Individual responses are given in Appendix 6.


Behavioural Assessments
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.


Functional Performance Tests
There were no toxicologically significant changes in functional performance.
Males treated with 350 mg/kg bw/day showed a statistically significant increase in hind limb grip strength whilst females from all treatment groups showed a statistically significant increase in fore limb grip strength. The statistically significant differences were confined to one out of the three tests and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.


Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.


Body Weight
Group mean body weights, body weight changes and standard deviations are given in Table 7 and Table 8 (statistically significant differences are indicated). Group mean body weight data are presented graphically in Figure 1 and Figure 2. Individual data are given in Appendix 7 and Appendix 8.
Males treated with 350 mg/kg bw/day showed a statistically significant (p<0.01) reduction in body weight gain during Week 3. Although statistical analysis did not reveal any significant intergroup differences, a slight reduction in body weight gain was also evident in these males during Week 4; resulting in a reduction in overall body weight gain.

No such effects were detected in females treated with 350 or 150 mg/kg bw/day or in animals of either sex treated with 75 mg/kg bw/day.
Males treated with 150 mg/kg bw/day showed a statistically significant (p<0.05) reduction in body weight gain during Week 3. In isolation and in the absence of associated changes or an effect on overall body weight development the intergroup difference was considered not to be of toxicological importance.


Food Consumption and Food Efficiency
Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Individual and group mean food consumptions for females following mating and during lactation are presented in Appendix 9.

Food efficiencies for males and females during the pre-mating phase are given in Table 10.

No adverse effect on food consumption or food efficiency was detected for treated males throughout the treatment period.

No adverse effect on food consumption or food efficiency was detected for females during the pre-pairing, gestation or lactation phases of the study.

Statistical analysis of gestation and lactation data revealed no significant differences.


Water Consumption
No adverse effect on water consumption was detected.


Laboratory Investigations
Haematology
Group mean values and standard deviations for test and control group animals are given in Table 17 (statistically significant differences are indicated). Individual data are given in Appendix 16 and Appendix 17.

There were no toxicologically significant effects detected in the haematological parameters examined.
Females from all treatment groups showed a statistically significant (p<0.05) increase in mean corpuscular volume. Females treated with 350 mg/kg bw/day also showed a statistically significant (p<0.01) increase in activated partial thromboplastin time. All individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered not to be of toxicological importance.


Blood Chemistry
Group mean values and standard deviations for test and control group animals are given in Table 18 (statistically significant differences are indicated). Individual data are given in Appendices 18 and 19.

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

Males treated with 350 and 150 mg/kg bw/day showed statistically significant increases (p<0.05) in creatinine and urea. Males treated with 350 mg/kg bw/day also showed statistically significant increases (p<0.05) in potassium concentration and total protein. The majority of individual values were within the normal ranges for rats of the strain and age used, and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

Females treated with 350 mg/kg bw/day showed statistically significant increases (p<0.05) in total protein and bilirubin. All individual values were within the normal ranges for rats of the strain and age used, and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance. Females treated with 350 mg/kg bw/day also showed statistically significant increases (p<0.05) in cholesterol and bile acid. The level of significance was minimal and in the absence of any histology correlates the intergroup difference was considered not to be of toxicological significance.


Pathology
Organ Weights
Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in Table 19 (statistically significant differences are indicated). Individual data are given in Appendix 20 and Appendix 21.

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

Males from all treatment groups showed a statistically significant reduction (p<0.05) in prostate weight both absolute and relative to terminal body weight. In the absence of a true dose related response or any histology correlates the intergroup differences were considered not to be of toxicological importance. Males treated with 350 and 150 mg/kg bw/day and females treated with 350 mg/kg bw/day showed statistically significant reductions in pituitary and thyroid weight both absolute and relative to terminal body weight. In the absence of any associated changes or histology correlates the intergroup differences were considered not to be of toxicological importance.


Necropsy
A summary incidence of necropsy findings is given in Tables 20 and 21. Individual data are given in Appendices 22 and 23.

Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Adults
The female treated with 350 mg/kg bw/day that was found dead on Day 41 showed distension in the stomach, caecum, duodenum, ileum and jejunum. The stomach, caecum, colon, duodenum, ileum and jejunum were also fluid filled.

No toxicologically significant macroscopic abnormalities were detected in males treated with 350 mg/kg bw/day, terminal kill females treated with 350 mg/kg bw/day or animals of either sex treated with 150 and 75 mg/kg bw/day.

One male treated with 350 mg/kg bw/day had pale lungs and two females treated with 75 mg/kg bw/day had reddened lungs at necropsy. In the absence of any treatment related histology correlates the macroscopic findings were considered to be within the range of normal background alterations.
One control male and one female treated with 350 mg/kg bw/day had increased pelvic space in the right kidney. In the absence of treatment in the case of the control male or any histology correlates the intergroup differences were considered of no toxicological importance.

The control female that was found dead had reddened lungs and gaseous distension in the caecum, ileum and jejunum.


Histopathology
A complete histopathology report is presented in Appendix 24.

Interim Deaths
The following treatment related microscopic findings were detected in the female treated with 350 mg/kg bw/day that was found dead on Day 41:
Stomach: Glandular stomach ulceration, focal (at the border region between the stomach and duodenum) and hyperkeratosis (mainly at the limiting ridge).
Esophagus: Hyperkeratosis/parakeratosis
Thymus: Atrophy (lymphocytolysis)
The control female that was found dead on Day 10 showed:
Esophagus: Degeneration/regeneration of the muscle fibers, focal (with focal destruction of muscle layer)
Trachea: Necrotizing inflammation
Lungs (with bronchi): Aspiration pneumonia
Accidental oesophageal injury and respiratory tract inflammation were considered the cause of the animal’s death.
The following treatment related microscopic findings were detected in terminal kill animals:

Stomach: Squamous cell hyperplasia and hyperkeratosis in forestomach and/or limiting ridge were evident in animals of either sex treated with 350 mg/kg bw/day. In addition, forestomach erosion was evident in one female treated with 350 mg/kg bw/day and hypertrophy of neck mucous cells in one male treated with 350 mg/kg bw/day was evident. All of these findings were considered to be the result of mild irritating properties of the test item.






Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 other: mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the higher incidence of clinical signs of salivation and respiratory pattern changes, a decreased body weight gain in males during Weeks 3 and 4 and signs of local irritation in the stomach that were observed at 350 mg/kg bw/day.
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
350 other: mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related effects were detected in the reproductive parameters examined

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulfonates to rats by gavage, at dose levels of 75, 150 and 350 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 350 mg/kg bw/day. No such effects were detected in animals of either sex treated with 150 or 75 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 150 mg/kg bw/day, based on the higher incidence of clinical signs of salivation and respiratory pattern changes, a decreased body weight gain in males during Weeks 3 and 4 and signs of local irritation in the stomach that were observed at 350 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 350 mg/kg bw/day.
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) and is 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 22 March 1996).

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 ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 75, 150 and 350 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioural 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 5 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 surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partumHaematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality. One female treated with 350 mg/kg bw/day was found dead on Day 41. This death was considered to be related to the microscopic observations detected for this animal.

There were no further treatment related deaths.

Clinical Observations. Animals of either sex treated with 350 and 150 mg/kg bw/day showed episodes of increased salivation throughout the treatment period. Incidences of noisy respiration were evident in animals of either sex treated with 350 mg/kg bw/day and in males treated with 150 mg/kg bw/day. One male treated with 350 mg/kg bw/day also showed a decreased respiratory rate on Day 18 and laboured respiration on Days 18 and 19. No such effects were detected in animals of either sex treated with 75 mg/kg bw/day.

Behavioural Assessment. There were no treatment-related changes in the behavioural 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. Males treated with 350 mg/kg bw/day showed a reduction in body weight gain during Weeks 3 and 4, subsequent overall body weight gain was also reduced in these males when compared to controls.  

No toxicologically significant effects were detected in females treated with 350 mg/kg bw/day or animals of either sex treated with 150 or 75 mg/kg bw/day.

Food Consumption and Food Efficiency. No adverse effect on food consumption was detected in treated animals.

Water Consumption. No adverse effect on water consumption was detected.

Laboratory Investigations:

Haematology. There were no toxicologically significant effects detected in the haematological parameters measured.

Blood Chemistry. There were no toxicologically significant effects detected in the blood chemical parameters measured.

Pathology:

Necropsy. The female treated with 350 mg/kg bw/day that was found dead on Day 41 had distension of the gastro intestinal tract. The gastro intestinal tract was also fluid filled.  

No toxicologically significant macroscopic abnormalities were detected in males treated with 350 mg/kg bw/day, terminal kill females treated with 350 mg/kg bw/day or animals of either sex treated with 150 and 75 mg/kg bw/day.

Organ Weights. No toxicologically significant effects were detected in the organ weights examined.

 

Histopathology:

Interim Deaths: The following treatment related microscopic findings were detected in the female treated with 350 mg/kg bw/day that was found dead on Day 41:

Stomach: Glandular stomach ulceration, focal (at the border region between the stomach and duodenum) and hyperkeratosis (mainly at the limiting ridge).

Esophagus: Hyperkeratosis/parakeratosis

Thymus: Atrophy (lymphocytolysis)

The following treatment related microscopic findings were detected in terminal kill animals:

Stomach: Squamous cell hyperplasia and hyperkeratosis in the forestomach and/or limiting ridge were recorded in animals of either sex treated with 350 mg/kg bw/day. In addition, forestomach erosion in one female treated with 350 mg/kg bw/day and hypertrophy of neck mucous cells in one male treated with 350 mg/kg bw/day was evident. 

Conclusion. The oral administration of 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulfonates to rats by gavage, at dose levels of 75, 150 and 350 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 350 mg/kg bw/day. No such effects were detected in animals of either sex treated with 150 or 75 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 150 mg/kg bw/day, based on the higher incidence of clinical signs of salivation and respiratory pattern changes, a decreased body weight gain in males during Weeks 3 and 4 and signs of local irritation in the stomach that were observed at 350 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 350 mg/kg bw/day.

Applicability of cross-reading.

This study is performed on salt of C10-13-alkylbenzene sulfonate with 3-(methacrylamidopropyl) trimethylammonium (MAPTA-ABS salt) rather than 3-(methacrylamidopropyl) dimethylammonium (MAPDA-ABS salt). Support for the read-across is separately attached to Chapter 13 of this IUCLID in the document “Justification in support of cross-reading between MAPDA-ABS salt and MAPTA-ABS salt”. The justification is build on comparable structure, the available information on toxicity of both salt parts ABS and MAPTA resp. MAPDA, and the confirmation obtained from the available data for both substances.