N,N,4-trimethylpiperazine-1-ethylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-25 to 2020-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-25 to 2020-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name: Jeffcat TAP, N,N,4-Trimethylpiperazine-1-ethylamine
- CAS number: 104-19-8
- Physical state/appearance: Clear colorless liquid
- Batch number: PFW160141
- Purity: 60-100%
- Label: Jeffcat TAP Lot no FW160141
- Expiry date: 30 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature and humidity in darkness; used/formulated: in light

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected 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 or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 11 weeks old
Females: approx. 14 weeks old
- Weight at study initiation:
Males: 283g - 355g
Females: 198g - 240g
- Fasting period before study: No
- Housing: 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.
Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum, A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.)
- Water: ad libitum, Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:
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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2017-09-04 To: 2017-11-07

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in Distilled water.
- The formulations were shown to be stable for at least 21 days when stored refrigerated. Formulations were therefore prepared every 2 weeks and stored at approximately 4ºC in the dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 0, 2, 5, 10 mg/mL
- Amount of vehicle (if gavage): 5mL/kg of distilled water. The volume of test and control item ad ministered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulations were shown to be stable for at least twenty-one days when stored refrigerated. F ormulations were therefore prepared fortnightly and stored at approximately 4ºC in the dark.
- Samples of the test item formulations were taken on three occasions and analyzed for concentration of the test substance

Duration of treatment / exposure:

Males : approx. 6 weeks
Females: up to 10 Weeks (including a two-week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate dose)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 4 (HIgh dose)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of previous toxicity work incl uding a Fourteen Day Repeated Dose Oral (Gavage) RangeFinding/Screening Toxicity Study in the Rat (Envigo study number: FL83TJ).
- Rationale for animal assignment (if not random): 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.
- Treatment volume: 5 mL/kg body weight.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing and one hour after dosing during the workweek (except for females during parturition where applicable).
- Parameters: overt signs of toxicity, ill-health, behavioral change.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 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, 7, 14 post partum and at terminal kill.

FOOD CONSUMPTION: Yes
- During the pre-pairing period : weekly food consumption was recorded for each cage of adults.
- For males after the mating phase: weekly food consumption was recorded for each cage of adults.
- 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 for the periods covering post partum Days 1-4, 4-7 and 7-14.

FOOD EFFICIENCY:
- 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 AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination or when necessary at termination
- Anaesthetic used for blood collection: not specified . Blood samples were obtained from the lateral tail vein.
- Animals fasted: no
- Number of animals: five males and five females selected from each test and control group
- Parameters checked : 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), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination or when necessary at termination
- Animals fasted: no
- Number of many animals: five males and five females selected from each test and control group
- Parameters checked: 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++).

OTHER:
FUNCTIONAL OBSERVATIONS
- prior to 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 for each animal using a purpose built arena
- parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation

FUNCTIONAL PERFORMANCE
- 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.
- ForelimbHindlimb 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.

SENSOR REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- Parameters observed : Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

THYROID HORMONE ANALYSIS
- serum and plasma samples were taken from all adult males and females at termination.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy.

Sperm parameters (parental animals):

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.

Litter observations:

LITTER DATA
- On completion of parturition (Day 0 post partum) the number of live and dead offspring was recorded.
- The following parameters were examined in F1 offspring:
Number of offspring born, Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum, Sex of offspring on Days 1, 4 and 13 post partum, Clinical condition of offspring from birth to Day 13 post partum, 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.

THYROID HORMONE ANALYSIS
- Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
- Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
Males: killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Females: killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS NECROPSY
- Post mortem procedures were performed on all adult animals and included any animal found dead or killed in extremis during the study.
- a full external and internal examination

ORGAN WEIGHT
- For five males and five females selected from each test and control group
- The following organs dissected free from fat and weighed before fixation : Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (weihed partially fixed), Prostate, Seminal vesicles (with coagulation gland), spleen, Testes, Thymus, Thyroid (weighed partially fixed with pa rathyroid), Uterus (weighed with cervix and oviducts)
- For all remaining animals, the following organs were weighed: Epididymides, Prostate, Seminal Vesicles (with coagulation gland), Ovaries, Pituitary (weighed after partial fixation), Thyroid (weighed after partial fixation with parathyroid), Uterus (weighed with Cervix).

HISTOPATHOLOGY
- from five males and five females selected from each test and control group had the following organs preserved in buffered 10% formalin.
- The tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination.
- Tissues / organs collected: Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and Bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Cowpers glands, Duodenum, Esophagus, Eyes (retained in Davidson's Fluid), Glans penis, Gross lesions, Heart, Ileum (including peyer's patches), Jejunum, Kidneys, LABC (levator anibulbocavernous) muscle, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillar y), Sciatic nerve, Seminal vesicles (with coagulation gland), Skin, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes (retained in Modified Davidson's Fluid), Thymus, Thyroid/ Parathyroid, Trachea, Urinary bladder, Uterus and Cervix (with oviducts), Vagina

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

GROSS NECROPSY
Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed or the offspring died during the study, then an additional internal examination was performed.

THYROID HORMONE ANALYSIS
-Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
-Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.

Statistics:

Homogeneity of variance from mean values was analyzed using Bartlett's test.
Intergroup variance was assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates.
Any transformed data were analyzed 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 is found, but the data showed non­homogeneity of means, the data was analyzed by a stepwise Dunnett's (parametric) or Steel (non-parametric) test to determine significant difference from the control group.
Where the data were unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett's test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett's test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann­ Whitney "U" test. Dose response relationships may also have been investigated by linear regression. Where the data was unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Reproductive indices:

Precoital interval: calculated as the time elapsing between initial pairing and the observation of positive evidence of mating
Mating index (%): (Number of animals mated / number of paired animals) x 100
Pregnancy index (%): (number of presumed pregnant animals / number of paired animals) x 100
Gestation length: calculated as the number of days of gestation includign the day for observation of mating and the start of parturition
Parturition index (%): (number of females delivering live offspring / number of pregnant females) x 100

Offspring viability indices:

Post - implantation loss (%): (number of implantations – total number of offspring born / Number of implantation sites) x 100
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1)x 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) x 100
% Male offspring (Sex Ratio): calculated for each litter value on days 1, 4, 7 and 13 post partum = (Number of male offspring/ Total number of offspring) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 10, 25 or 50 mg/kg bw/day.
- One female treated with 50 mg/kg bw/day showed increased post-dose salivation on a single occas ion (Day 55).
- Isolated incidences of noisy respiration were evident in either sex throughout the treatment groups.
- At these low frequencies and distributions, this is considered to indicate possible difficulties in dosing particular animals on isolated occasions and not indicative of systemic toxicity.
- Incidental observations, unrelated to test item administration, included one control male with staini ng around the eyes on an isolated occasion (Day 31) and one female treated with 10 mg/kg bw/day showed a period of generalized fur loss (Days 55-61).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no toxicologically significant effect on body weight or body weight gains for males at 10, 25 or 50 mg/kg bw/day. Males treated with 50 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weig ht gain during Week 5, however, an increase in body weight gain is considered not to be an adverse effect of treatment. There was no toxicologically significant effect on body weight or body weight gains for females during pre-pairing at 10, 25 or 50 mg/kg bw/day, or for females during gestation and lactation at 10 or 25 mg/ kg bw/day. At 50 mg/kg bw/day, females showed lower body weights throughout gestation, attaining statistical significance on Days 7 (p<0.05) and 14 (p<0.01). Overall body weight gains throughout gestation were also statistically significantly lower between Days 0-14 (p<0.05) and Days 0-20 (p<0.01). Lower body weights continued throughout lactation for females at 50 mg/kg bw/day, attaining statistical si gnificance on Days 4 (p<0.01), 7 (p<0.01), and 14 (p<0.05). Body weight gain between Days 1 and 4 of lactation and overall body weight gains between Days 1 and 7 and Days 1 and 14 of lactation were lower than controls, but did not attain statistical significance. For more information, please refer to Table 2 and 3.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

-There was no effect of treatment on food consumption or food conversion efficiency for males or females during pre-pairing at 10, 25 or 50 mg/kg bw/day.
-Females treated at 50 mg/kg bw/day: statistically significant reduction (p<0.05) in food consumption during the first week of gestation, but noticeable improvement afterwards. Between Days 4 and 7 and Days 7 and 14 of lactation food consumption were lower than controls, but values did not attain statistical significance.
-There was no effect of treatment on food consumption for females at 10 and 25 mg/kg bw/day during gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food conversion efficiency for males or females during pre-pairing at 10, 25 or 50 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on water consumption for either sex at 10, 25 or 50 mg/kg bw/day; daily visual inspection of water bottles revealed no overt intergroup differences

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of the hematology parameters measured did not indicate any obvious effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day. Males treated with 25 and 50 mg/kg bw/day showed a statistically significant increase in hemoglobin levels (p<0.05). All individual values were within the historical control range, whereas, one individual control value was below the historical control range. The intergroup differences did not show a true dose related response and there was no concurrent effects on hematocrit or red blood cell values in these animals, which usually reflect any abnormalities in hemoglobin levels. These findings were therefore considered not to be of toxicological significance.
Males treated with 25 and 50 mg/kg bw/day also showed a statistically significant increase (p<0.05 and p<0.01 respectively) in reticulocytes. Although all of the individual values for 50 mg/kg bw/day males were above the historical control range, all but one individual value for the 25 mg/kg bw/day males were within the historical control range. One control individual value was also below the histor ical control range which may have impacted the overall intergroup difference. There was no evidenc e of red blood cell destruction or anemia which could have triggered the increased reticulocyte pro duction and no corresponding histopathological changes were evident in the bone marrow. The intergroup differences were therefore considered not to be of toxicological significance. Males treated at 50 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total leukoc yte count and males from all treatment groups showed a statistically significant reduction (p<0.05, p<0.05 and p<0.01 at dose levels of 10, 25 and 50 mg/kg bw/day respectively) in lymphocytes. All of the individual values for total leukocyte count were within the historical control range and the majority of individual values for lymphocytes were also within the historical control range. A true dose related response was not observed for either parameter, and in the absence of any associated histopat hological correlates, the intergroup differences were considered not to be of toxicological significance. All treated females showed a statistically significant increase (p<0.05) in reticulocytes. A true dose related response was not evident, the majority of individual values were within the historical control range and there was no associated histopathological correlates. The intergroup difference was therefore considered not to be of toxicological significance.
For more information, please refer to Table 4.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemical parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day. Males treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.05) in glucose levels whilst females from this treatment group showed a statistically significant increase (p<0.05) in albumin levels. All of the individual values were within historical control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance. Bilirubin was statistically significantly increased (p<0.05) in all treated males. However, all individual values were within the historical control range, a true dose related response was not evident and no corresponding histopathological changes were evident in the liver. Therefore, the intergroup differences were considered of no toxicological significance.
For more information, please refer to Table 5.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

BEHAVIORAL ASSESSMENTS
Behavioral assessments did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day. Incidences of noisy respiration were evident in one male from the control group and one male from ea ch treatment group at some time between Weeks 3 and 5. One female treated with 25 mg/kg bw/day and one female treated with 50 mg/kg bw/day showed noisy respiration during Week 2 of treatment. The female treated with 25 mg/kg bw/day continued to show noisy respiration on the assessments days throughout gestation. These observations are likely to reflect possible dosing difficulties on these occasions rather than true systemic toxicity and correlate with the daily clinical observations recorded.

FUNCTIONAL PERFORMANCE TESTS
Functional performance tests did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/ kg bw/day. Motor activity assessment did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day. Overall activity was statistically significantly higher (p<0.05) for all treated females. However, all individual values, with the exception of one female treated with 25 mg/kg bw/day were within the historical control range and a true dose-related response was not evident. In the absence of a similar effect in males or any clinical signs to suggest a neurotoxic effect, it is likely that this finding is due to normal biological variation. Therefore, this finding is due to normal biological variation.

SENSORY REACTIVITY
Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.
For more information, please refer to table 1.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Brain: vacuolation/hypertrophy of the choroid plexus was present in two males and all females treate d with 50 mg/kg bw/day. It occurred in all brain regions where choroid plexus was present but was r ecorded under cerebrum for clarity. No such effects were evident in animals of either sex treated with 25 or 10 mg/kg bw/day.
Respiratory Tract: vacuolation was present in the tracheal epithelium of three males and three females treated with 50 mg/kg bw/day. Vacuolation was present in epithelium lining the bronchi of all males and females treated with 50 mg/kg bw/day. No such effects were evident in animals of either sex treated with 25 or 10 mg/kg bw/day.
Testes: vacuolation was present at a minimal severity in the tubular epithelium of eight males treated with 50 mg/kg bw/day. Minimal tubular degeneration, multifocal and bilateral in nature, was noted in two males treated with 50 mg/kg bw/day and is considered treatmentrelated. There was no apparent effect on the reproductive performance, however, rats have a high capacity for sperm production.
Focal unilateral tubular degeneration was present in some males treated with 25 mg/kg bw/day, however, this was considered to be a background change in view of its unilateral and focal nature. No such effects were evident in males treated with 10 mg/kg bw/day. There were no test item-related microscopic findings in the reproductive tracts following the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analysis: Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 50 mg/kg bw/day. Statistical analysis of the data did reveal a statistically significant increase (p<0.05) in T4 values in female offspring from adult females treated with 50 mg/kg bw/day. However, in the absence of a similar effect in male offspring or adult males the intergroup difference was considered not to be of toxicological significance

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 10, 25 or 50 mg/kg bw/day, with all females showing regular cycling.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation was present at a minimal severity in the tubular epithelium of eight males treated with 50 mg/kg bw/day. Minimal tubular degeneration, multifocal and bilateral in nature, was noted in two males treated with 50 mg/kg bw/day and is considered treatmentrelated. There was no apparent effect on the reproductive performance, however, rats have a high capacity for sperm production.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating: Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 10, 25 or 50 mg/kg bw/day.
- Fertility: There was no effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 10, 25 or 50 mg/kg bw/day. All pairings produced a successful pregnancy.
- Gestation Length: The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 10, 25 or 50 mg/kg bw/day.
- Litter responses: All females gave birth to a live litter and successfully reared young to termination at Day 13 of age.

The primary observation in the study was vacuolation in the brain and respiratory tract of males and females treated at 50 mg/kg bw/day (high dose group) and testes of males treated at 50 mg/kg bw/day. All other parameters measured or observed showed no test item-related effects. Regarding vacuolation in the brain, respiratory tract and testes, the vacuoles observed are considered transient and non-adverse based on scientific peer-reviewed literature regarding weakly basic aliphatic amines and supporting evidence from similar compounds within the same chemical family of aliphatic amines. The observation of vacuolation is considered to be transient and non-adverse for the test substance , and as such the effects are of minimal toxicological importance. Since no adverse effects have been identified at 50 mg/kg bw/day or below this dose level for either sex, the NOAEL for systemic toxicity is established at 50 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Any observations were considered to be non-adverse (justification in 'details on results')

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Any observations were considered to be non-adverse (justification in 'details on results')

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of maternal treatment on offspring survival to Day 13 of age at dosages of 10, 25 or 50 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 50 mg/kg bw/day.
Statistical analysis of the data did reveal a statistically significant increase (p<0.05) in T4 values in female offspring from adult females treated with 50 mg/kg bw/day. However, in the absence of a similar effect in male offspring or adult males the intergroup difference was considered not to be of toxicological significance.
For more information, please refer to table 6.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 10, 25 or 50 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analysis: Statistical analysis of the data did reveal a statistically significant increase (p<0.05) in T4 values in female offspring from adult females treated with 50 mg/kg bw/day. However, in the absence of a similar effect in male offspring or adult males the intergroup difference was considered not to be of toxicological significance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

-There was no effect of maternal treatment on the number of implantations, post-implantation loss and live birth index, and subsequent offspring survival to Day 13 of age at dosages of 10, 25 or 50 mg/kg bw/day. Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Statistical analysis of the data did not reveal any significant intergroup differences.
-There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, visible nipple count in male offspring on Day 13 post partum, or anogenital distance at 10, 25 or 50 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Analytical verification

The results indicate that the prepared formulations were within 91-103% of the nominal concentration.

Summary of the results from the 14day repeated dose oral (gavage) range-finding toxicity study

The test substance was administered in the rat for 14 consecutive days. In this study, at dose levels of 100 and 200 mg/kg bw/day, treatment-related effects were evident in animals of both sexes.  The necessary early termination or reduction in body weight gain and reduction in food consumption detected at 200 and 100 mg/kg bw/day were sufficient to exclude these dose levels from further investigation.  Over a period of fourteen days, the effect on body weight development at 50 mg/kg bw/day was considered not to represent a significantly adverse effect of treatment and was considered a suitable high dose level for subsequent studies.

Details on results

Table 1: Functional test: Overall activity

Sex (dose-mg/kg bw/day)	Males(0)	Males(10)	Males(25)	Males(50)	Females(0)	Females(10)	Females(25)	Females(50)
Overall Activity (Mean +/-SD)	398 +/- 86.0	411 +/- 51.1	355 +/-117.2	388 +/- 113.6	253 +/- 120.2	418* +/-60.8	433.6* +/-107.1	385.4* +/-65.6

Probability values (p) are presented as follows: ** p<0.01, * p<0.05, p>0.05 (not significant)

Historical control data: - for males: range (mean+/-2 sd) 153 -459 secs ; mean +/-sd 306 +/-77 - for females: range (mean +/-2 sd) 94 -522 secs; mean +/-sd 308 +/-107

Table 2: Group Mean Body Weight Values of Females during Gestation

Days  (dose-mg/kg bw/day)	0	7	14	20
0	236.3 +/-11.6	261.8 +/- 10.3	290.8 +/-12.4	348.8 +/-32.6
10	227.0 +/-12.0	252.0 +/- 11.2	279.0 +/-14.4	338.9 +/-20.1
25	229.8 +/-14.4	256.9 +/-17.6	284.5 +/-19.8	345.1 +/-27.1
50	225.6 +/-9.3	246.1*+/-10.4	270.8**+/-12.3	325.4 +/-18.0

Probability values (p) are presented as follows: ** p<0.01, * p<0.05, p>0.05 (not significant)

Table 3: Group Mean Body Weight Values of Females during Lactation

Days  (dose-mg/kg bw/day)	1	4	7	14
0	264.1 +/-17.5	282.5 +/-14.5	292.5 +/-15.5	319.3 +/-16.7
10	249.5 +/-17.9	269.0 +/-15.7	281.3 +/-12.5	306.3 +/-15.6
25	258.0 +/-23.1	277.1 +/-21.3	284.0 +/-23.5	312.8 +/-24.5
50	246.3 +/-12.7	259.5**+/-10.8	267.5**+/-11.1	297.0*+/-12.2

Probability values (p) are presented as follows: ** p<0.01, * p<0.05, p>0.05 (not significant)

Table 4: Group Mean Hematological Value

Sex (dose-mg/kg bw/day)	Males(0)	Males(10)	Males(25)	Males(50)	Females(0)	Females(10)	Females(25)	Females(50)
Hemoglobin (g/dL) (Mean +/- SD)	15.52 +/-0.66	14.92 +/-1.95	16.44* +/-0.38	16.42* +/-0.52	14.08 +/-0.29	14.34 +/-0.49	14.52 +/-0.88	14.86 +/-0.39
Leukocytes (10^9/L) (Mean +/- SD)	8.50 +/-0.95	6.92 +/-1.57	7.50 +/-1.54	6.24* +/-1.01	5.52 +/-1.14	4.94 +/-0.63	6.28 +/-2.68	4.94 +/-1.61
Lymphocytes(10^9/L) (Mean +/- SD)	5.60 +/-0.738	4.216* +/-0.938	4.374* +/-0.875	3.826** +/-0.604	3.342 +/-0.707	2.778 +/-0.340	3.936 +/-1.676	2.860 +/-0.975
Reticulocytes (%) (Mean +/- SD)	3.84 +/-0.52	4.42 +/-0.75	4.60* +/- 0.46	5.68** +/-0.28	4.22 +/-0.44	5.28* +/-0.76	4.70* +/-0.70	5.12* +/-0.38

Probability values (p) are presented as follows:** p<0.01, * p<0.05, p>0.05 (not significant)

Historical control data:

	males				females
	range (mean +/-2sd)	mean	sd	no.	range (mean +/-2sd)	mean	sd	no.
Hb (g/dL)	14.8 -18.3	16.6	0.9	85	12.4 -16.0	14.2	0.9	87
Leukocytes (10^9/L)	4.8 -10.8	7.8	1.5	86	3.4 -8.2	5.8	1.2	88
Lymphocytes (10^9/L)	3.8 -8.96	6.38	1.29	85	1.99 -6.14	4.07	1.04	89
reticulocytes (%)	3.1 -4.6	3.8	0.4	83	3.0 -5.3	4.2	0.6	84

Table 5: Group Mean Blood Chemical Values

Sex (dose-mg/kg bw/day)	Males(0)	Males(10)	Males(25)	Males(50)	Females(0)	Females(10)	Females(25)	Females(50)
Glucose (mg/dL)	171.6 +/-15.0	161.4 +/-7.9	164.2 +/-19.1	149.4* +/-8.2	139.6 +/-13.7	138.0 +/-8.6	141.4 +/-25.3	143.2 +/-12.7
Albumin (d/dL)	3.34 +/-0.27	3.58 +/-0.41	3.72 +/-0.13	3.68 +/-0.11	3.12 +/-0.11	3.26 +/-0.11	3.18 +/-0.08	3.38* +/-0.22
Bilirubin (mg/dL)	0.056 +/-0.021	0.092* +/-0.031	0.110* +/-0.025	0.096* +/-0.022	0.080 +/-0.021	0.080 +/-0.024	0.074 +/-0.021	0.080 +/-0.019

Probability values (p) are presented as follows:** p<0.01, * p<0.05, p>0.05 (not significant)

Historical control data:

	males				females
	range (mean +/- 2 sd)	mean	sd	no.	range (mean +/- 2 sd)	mean	sd	no.
glucose (mg/dL)	116 -211	163	24	84	98 -175	137	19	86
albumin (g/dL)	2.9 -4.3	3.6	0.3	86	2.7 -3.7	3.2	0.2	85
bilirubin (mg/dL)	0.02 -0.16	0.09	0.04	84	0.01 -0.14	0.08	0.03	87

Table 6 : Mean Serum T4 Concentrations (pg/mL)

Sex - Ages  (dose-mg/kg bw/day)	Males-Adults terminal	Male offsprings-13 days	Female offsprings-13 days
0	37900 +/-6900	37500 +/-7800	38800 +/-4820
10	40400 +/-7550	39500 +/-8270	34900 +/-5220
25	46300 +/-9950	43000 +/-5400	41700 +/-5460
50	41800 +/-7590	42900 +/-7550	44200* +/-5650

Probability values (p) are presented as follows: ** p<0.01, * p<0.05, p>0.05 (not significant)

Conclusions:

The oral administration of the test substance to rats by gavage, at dose levels of 10, 25 and 50 mg/kg bw/day, resulted in microscopic effects in animals of either sex treated with 50 mg/kg bw/day, which were considered to be transient and non-adverse, and as such the effects are of minimal toxicological importance. Since no adverse effects have been identified at 50 mg/kg bw/day or below this dose level for either sex, the NOAEL for systemic toxicity is established at 50 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 50 mg/kg bw/day. The test substance is considered not classified as reproductive toxicant.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2020

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N,4-trimethylpiperazine-1-ethylamine

EC Number:

203-183-7

EC Name:

N,N,4-trimethylpiperazine-1-ethylamine

Cas Number:

104-19-8

Molecular formula:

C9H21N3

IUPAC Name:

dimethyl[2-(4-methylpiperazin-1-yl)ethyl]amine

Test material form:

liquid

Specific details on test material used for the study:

- Name: Jeffcat TAP, N,N,4-Trimethylpiperazine-1-ethylamine
- CAS number: 104-19-8
- Physical state/appearance: Clear colorless liquid
- Batch number: PFW160141
- Purity: 60-100%
- Label: Jeffcat TAP Lot no FW160141
- Expiry date: 30 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature and humidity in darkness; used/formulated: in light

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected 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 or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 11 weeks old
Females: approx. 14 weeks old
- Weight at start of treatment:
Males: 283g - 355g
Females: 198g - 240g
- Fasting period before study: No
- Housing:
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.
- Diet: ad libitum, a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.)
- Water: ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:
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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2017-09-04 To: 2017-11-07

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
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 toxicity of the test item to man.

Vehicle:

water

Remarks:

Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in Distilled water.
- The formulations were shown to be stable for at least 21 days when stored refrigerated. Formulations were therefore prepared every 2 weeks and stored at approximately 4ºC in the dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 0, 2, 5, 10 mg/mL
- Amount of vehicle (if gavage): 5mL/kg of distilled water. 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.
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulations were shown to be stable for at least twenty-one days when stored refrigerated. Formulations were therefore prepared fortnightly and stored at approximately 4ºC in the dark.
- Samples of the test item formulations were taken on three occasions and analyzed for concentration of the test substance

Duration of treatment / exposure:

Males : approx. 6 weeks
Females: up to 10 Weeks (including a two-week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate dose)

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) RangeFinding/Screening Toxicity Study in the Rat (Envigo study number: FL83TJ).
- Rationale for animal assignment (if not random): 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.
- Treatment volume: 5 mL/kg body weight

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing and one hour after dosing during the workweek (except for females during parturition where applicable).
- Parameters: overt signs of toxicity, ill-health, behavioral change.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 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, 7, 14 post partum and at terminal kill.

FOOD CONSUMPTION: Yes
- During the pre-pairing period : weekly food consumption was recorded for each cage of adults.
- For males after the mating phase: weekly food consumption was recorded for each cage of adults.
- 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 for the periods covering post partum Days 1-4, 4-7 and 7-14.

FOOD EFFICIENCY:
- 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 AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination or when necessary at termination
- Anaesthetic used for blood collection: not specified . Blood samples were obtained from the lateral tail vein.
- Animals fasted: no
- Number of animals: five males and five females selected from each test and control group
- Parameters checked : 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), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination or when necessary at termination
- Animals fasted: no
- Number of many animals: five males and five females selected from each test and control group
- Parameters checked: 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++).

OTHER:
FUNCTIONAL OBSERVATIONS
- prior to 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 for each animal using a purpose built arena
- parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation

FUNCTIONAL PERFORMANCE
- 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.
- ForelimbHindlimb 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.

SENSOR REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- Parameters observed : Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

THYROID HORMONE ANALYSIS
- serum and plasma samples were taken from all adult males and females at termination.

Sacrifice and pathology:

SACRIFICE
Males: killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Females: killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS NECROPSY
- Post mortem procedures were performed on all adult animals and included any animal found dead or killed in extremis during the study.
- a full external and internal examination

ORGAN WEIGHT
- For five males and five females selected from each test and control group
The following organs dissected free from fat and weighed before fixation : Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (weihed partially fixed), Prostate, Seminal vesicles (with coagulation gland), spleen, Testes, Thymus, Thyroid (weighed partially fixed with parathyroid), Uterus (weighed with cervix and oviducts)
- For all remaining animals, the following organs were weighed: Epididymides, Prostate, Seminal Vesicles (with coagulation gland), Ovaries, Pituitary (weighed after partial fixation), Thyroid (weighed after partial fixation with parathyroid), Uterus (weighed with Cervix).

HISTOPATHOLOGY
- from five males and five females selected from each test and control group had the following organs preserved in buffered 10% formalin.
- The tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination.
- Tissues / organs collected: Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and Bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Cowpers glands, Duodenum, Esophagus, Eyes (retained in Davidson's Fluid), Glans penis, Gross lesions, Heart, Ileum (including peyer's patches), Jejunum, Kidneys, LABC (levator ani-bulbocavernous) muscle, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulation gland), Skin, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes (retained in Modified Davidson's Fluid), Thymus, Thyroid/Parathyroid, Trachea, Urinary bladder, Uterus and Cervix (with oviducts), Vagina

Statistics:

Homogeneity of variance from mean values was analyzed using Bartlett's test.
Intergroup variance was assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates.
Any transformed data were analyzed 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 is found, but the data showed non­ homogeneity of means, the data was analyzed by a stepwise Dunnett's (parametric) or Steel (non-parametric) test to determine significant difference from the control group.
Where the data were unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett's test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett's test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann­ Whitney "U" test. Dose response relationships may also have been investigated by linear regression. Where the data was unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

-There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 10, 25 or 50 mg/kg bw/day.
- One female treated with 50 mg/kg bw/day showed increased post-dose salivation on a single occasion (Day 55).
- Isolated incidences of noisy respiration were evident in either sex throughout the treatment groups.
- At these low frequencies and distributions, this is considered to indicate possible difficulties in dosing particular animals on isolated occasions and not indicative of systemic toxicity.
- Incidental observations, unrelated to test item administration, included one control male with staining around the eyes on an isolated occasion (Day 31) and one female treated with 10 mg/kg bw/day showed a period of generalized fur loss (Days 55-61).

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no toxicologically significant effect on body weight or body weight gains for males at 10, 25 or 50 mg/kg bw/day.
Males treated with 50 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during Week 5, however, an increase in body weight gain is considered not to be an adverse effect of treatment.
There was no toxicologically significant effect on body weight or body weight gains for females during pre-pairing at 10, 25 or 50 mg/kg bw/day, or for females during gestation and lactation at 10 or 25 mg/kg bw/day.
At 50 mg/kg bw/day, females showed lower body weights throughout gestation, attaining statistical significance on Days 7 (p<0.05) and 14 (p<0.01). Overall body weight gains throughout gestation were also statistically significantly lower between Days 0-14 (p<0.05) and Days 0-20 (p<0.01). Lower body weights continued throughout lactation for females at 50 mg/kg bw/day, attaining statistical significance on Days 4 (p<0.01), 7 (p<0.01), and 14 (p<0.05). Body weight gain between Days 1 and 4 of lactation and overall body weight gains between Days 1 and 7 and Days 1 and 14 of lactation were lower than controls, but did not attain statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on food consumption or food conversion efficiency for males or females during pre-pairing at 10, 25 or 50 mg/kg bw/day.
-Females treated at 50 mg/kg bw/day: statistically significant reduction (p<0.05) in food consumption during the first week of gestation, but noticeable improvement afterwards. Between Days 4 and 7 and Days 7 and 14 of lactation food consumption were lower than controls, but values did not attain statistical significance.
There was no effect of treatment on food consumption for females at 10 and 25 mg/kg bw/day during gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food conversion efficiency for males or females during pre-pairing at 10, 25 or 50 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on water consumption for either sex at 10, 25 or 50 mg/kg bw/day; daily visual inspection of water bottles revealed no overt intergroup differences

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of the hematology parameters measured did not indicate any obvious effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.
Males treated with 25 and 50 mg/kg bw/day showed a statistically significant increase in hemoglobin levels (p<0.05). All individual values were within the historical control range, whereas, one individual control value was below the historical control range. The intergroup differences did not show a true dose related response and there was no concurrent effects on hematocrit or red blood cell values in these animals, which usually reflect any abnormalities in hemoglobin levels. These findings were therefore considered not to be of toxicological significance.
Males treated with 25 and 50 mg/kg bw/day also showed a statistically significant increase (p<0.05 and p<0.01 respectively) in reticulocytes. Although all of the individual values for 50 mg/kg bw/day males were above the historical control range, all but one individual value for the 25 mg/kg bw/day males were within the historical control range. One control individual value was also below the historical control range which may have impacted the overall intergroup difference. There was no evidence of red blood cell destruction or anemia which could have triggered the increased reticulocyte production and no corresponding histopathological changes were evident in the bone marrow. The intergroup differences were therefore considered not to be of toxicological significance.
Males treated at 50 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total leukocyte count and males from all treatment groups showed a statistically significant reduction (p<0.05, p<0.05 and p<0.01 at dose levels of 10, 25 and 50 mg/kg bw/day respectively) in lymphocytes. All of the individual values for total leukocyte count were within the historical control range and the majority of individual values for lymphocytes were also within the historical control range. A true dose related response was not observed for either parameter, and in the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
All treated females showed a statistically significant increase (p<0.05) in reticulocytes. A true dose related response was not evident, the majority of individual values were within the historical control range and there was no associated histopathological correlates. The intergroup difference was therefore considered not to be of toxicological significance.
For more information, please refer to Table 2.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemical parameters did not indicate any obvious effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.
Males treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.05) in glucose levels whilst females from this treatment group showed a statistically significant increase (p<0.05) in albumin levels. All of the individual values were within historical control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance. Bilirubin was statistically significantly increased (p<0.05) in all treated males. However, all individual values were within the historical control range, a true dose related response was not evident and no corresponding histopathological changes were evident in the liver. Therefore, the intergroup differences were considered of no toxicological significance.
For more information, please refer to Table 3.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

BEHAVIORAL ASSESSMENTS
Behavioral assessments did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.
Incidences of noisy respiration were evident in one male from the control group and one male from each treatment group at some time between Weeks 3 and 5. One female treated with 25 mg/kg bw/day and one female treated with 50 mg/kg bw/day showed noisy respiration during Week 2 of treatment. The female treated with 25 mg/kg bw/day continued to show noisy respiration on the assessments days throughout gestation. These observations are likely to reflect possible dosing difficulties on these occasions rather than true systemic toxicity and correlate with the daily clinical observations recorded.

FUNCTIONAL PERFORMANCE TESTS
Functional performance tests did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.
Motor activity assessment did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.
Overall activity was statistically significantly higher (p<0.05) for all treated females. However, all individual values, with the exception of one female treated with 25 mg/kg bw/day were within the historical control range and a true dose-related response was not evident. In the absence of a similar effect in males or any clinical signs to suggest a neurotoxic effect, it is likely that this finding is due to normal biological variation.
Therefore, this finding is due to normal biological variation.

SENSORY REACTIVITY
Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 10, 25 or 50 mg/kg bw/day.

For more information, please refer to Table 1.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no effect of treatment on organ weights for either sex at dosages of 10, 25 or 50 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic abnormalities detected at dosages of 10, 25 or 50 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Brain: vacuolation/hypertrophy of the choroid plexus was present in two males and all females treated with 50 mg/kg bw/day. It occurred in all brain regions where choroid plexus was present but was recorded under cerebrum for clarity.
No such effects were evident in animals of either sex treated with 25 or 10 mg/kg bw/day.
Respiratory Tract: vacuolation was present in the tracheal epithelium of three males and three females treated with 50 mg/kg bw/day. Vacuolation was present in epithelium lining the bronchi of all males and females treated with 50 mg/kg bw/day. No such effects were evident in animals of either sex treated with 25 or 10 mg/kg bw/day.
Testes: vacuolation was present at a minimal severity in the tubular epithelium of eight males treated with 50 mg/kg bw/day. Minimal tubular degeneration, multifocal and bilateral in nature, was noted in two males treated with 50 mg/kg bw/day and is considered treatment-related. There was no apparent effect on the reproductive performance, however, rats have a high capacity for sperm production.
Focal unilateral tubular degeneration was present in some males treated with 25 mg/kg bw/day, however, this was considered to be a background change in view of its unilateral and focal nature. No such effects were evident in males treated with 10 mg/kg bw/day.
There were no test item-related microscopic findings in the reproductive tracts following the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analysis: Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 25 or 50 mg/kg bw/day. Statistical analysis of the data did reveal a statistically significant increase (p<0.05) in T4 values in female offspring from adult females treated with 50 mg/kg bw/day. However, in the absence of a similar effect in male offspring or adult males the intergroup difference was considered not to be of toxicological significance.

Details on results:

Based on the proposed mechanism of action of osmotic effects and the observation of transient vacuolization in other aliphatic amines, the observation of vacuolization is considered to be transient and non-adverse and as such the effects are of minimal toxicological importance. This description does not meet the criteria for consideration of STOT RE classification as stated in the ECHA Guidance on the application of the CLP Criteria. Therefore, this effect does not indicate significant toxicity in relation to STOT RE classification in GHS. Since no adverse effects have been identified at 50 mg/kg bw/day or below this dose level for either sex, the NOAEL for systemic toxicity is set at 50 mg/kg bw/day.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Observation of vacuolation is considered to be non-adverse (justification in 'details on results')

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Analytical verification

The results indicate that the prepared formulations were within 91-103% of the nominal concentration.

Summary of the results from the 14day repeated dose oral (gavage) range-finding toxicity study

The test substance was administered in the rat for 14 consecutive days. In this study, at dose levels of 100 and 200 mg/kg bw/day, treatment-related effects were evident in animals of both sexes.  The necessary early termination or reduction in body weight gain and reduction in food consumption detected at 200 and 100 mg/kg bw/day were sufficient to exclude these dose levels from further investigation.  Over a period of fourteen days, the effect on body weight development at 50 mg/kg bw/day was considered not to represent a significantly adverse effect of treatment and was considered a suitable high dose level for subsequent studies.

Details on results

Table 1: Functional test: Overall activity

Sex (dose in mg/kg bw/day)	Males (0)	Males (10)	Males (25)	Males (50)	Females (0)	Females (10)	Females (25)	Females (50)
Overall Activity (Mean +/-SD)	398.4 +/- 86.0	411.4 +/- 51.1	355.0 +/-117.2	388.2 +/- 113.6	253.0 +/- 120.2	418.8* +/-60.8	433.6* +/-107.1	385.4* +/-65.6

Probability values (p) are presented as follows: ** p<0.01, * p<0.05, p>0.05 (not significant)

Historical control data:

- for males: range (mean+/-2 sd) 153 -459 secs ; mean +/-sd 306 +/-77

- for females: range (mean +/-2 sd) 94 -522 secs; mean +/-sd 308 +/-107

Table 2: Group Mean Hematological Value

Sex (dose-mg/kg bw/day)	Males (0)	Males (10)	Males (25)	Males (50)	Females (0)	Females (10)	Females (25)	Females (50)
Hemoglobin (g/dL) (Mean +/- SD)	15.52 +/-0.66	14.92 +/-1.95	16.44* +/-0.38	16.42* +/-0.52	14.08 +/-0.29	14.34 +/-0.49	14.52 +/-0.88	14.86 +/-0.39
Leukocytes (10^9/L) (Mean +/- SD)	8.50 +/-0.95	6.92 +/-1.57	7.50 +/-1.54	6.24* +/-1.01	5.52 +/-1.14	4.94 +/-0.63	6.28 +/-2.68	4.94 +/-1.61
Lymphocytes(10^9/L) (Mean +/- SD)	5.60 +/-0.738	4.216* +/-0.938	4.374* +/-0.875	3.826** +/-0.604	3.342 +/-0.707	2.778 +/-0.340	3.936 +/-1.676	2.860 +/-0.975
Reticulocytes (%) (Mean +/- SD)	3.84 +/-0.52	4.42 +/-0.75	4.60* +/- 0.46	5.68** +/-0.28	4.22 +/-0.44	5.28* +/-0.76	4.70* +/-0.70	5.12* +/-0.38

Probability values (p) are presented as follows: ** p<0.01, * p<0.05, p>0.05 (not significant)

Historical control data:

	males				females
	range (mean +/-2sd)	mean	sd	no.	range (mean +/-2sd)	mean	sd	no.
Hb (g/dL)	14.8 -18.3	16.6	0.9	85	12.4 -16.0	14.2	0.9	87
Leukocytes (10^9/L)	4.8 -10.8	7.8	1.5	86	3.4 -8.2	5.8	1.2	88
Lymphocytes (10^9/L)	3.8 -8.96	6.38	1.29	85	1.99 -6.14	4.07	1.04	89
reticulocytes (%)	3.1 -4.6	3.8	0.4	83	3.0 -5.3	4.2	0.6	84

Table 3: Group Mean Blood Chemical Values

Sex (dose-mg/kg bw/day)	Males(0)	Males(10)	Males(25)	Males(50)	Females(0)	Females(10)	Females(25)	Females(50)
Glucose (mg/dL)	171.6 +/-15.0	161.4 +/-7.9	164.2 +/-19.1	149.4* +/-8.2	139.6 +/-13.7	138.0 +/-8.6	141.4 +/-25.3	143.2 +/-12.7
Albumin (g/dL)	3.34 +/-0.27	3.58 +/-0.41	3.72 +/-0.13	3.68 +/-0.11	3.12 +/-0.11	3.26 +/-0.11	3.18 +/-0.08	3.38* +/-0.22
Bilirubin (mg/dL)	0.056 +/-0.021	0.092* +/-0.031	0.110* +/-0.025	0.096* +/-0.022	0.080 +/-0.021	0.080 +/-0.024	0.074 +/-0.021	0.080 +/-0.019

Probability values (p) are presented as follows:** p<0.01, * p<0.05, p>0.05 (not significant)

Historical control data:

	males				females
	range (mean+/-2sd)	mean	sd	no.	range (mean+/-2sd)	mean	sd	no.
Glucose (mg/dL)	116 -211	163	24	84	98 -175	137	19	86
Albumin (g/dL)	2.9 -4.3	3.6	0.3	86	2.7 -3.7	3.2	0.2	85
Bilirubin (mg/dL)	0.02 -0.16	0.09	0.04	84	0.01 -0.14	0.08	0.03	87

Applicant's summary and conclusion

Conclusions:

The oral administration of Jeffcat TAP to rats by gavage, at dose levels of 10, 25 and 50 mg/kg bw/day, resulted in test article related microscopic effects in animals of either sex treated with 50 mg/kg bw/day. No such effects were evident in animals of either sex treated with 25 or 10 mg/kg bw/day. However, Based on the proposed mechanism of action of osmotic effects and the observation of transient vacuolization in other aliphatic amines, the observation of vacuolization is considered to be transient and non-adverse and as such the effects are of minimal toxicological importance. This description does not meet the criteria for consideration of STOT RE classification as stated in the ECHA Guidance on the application of the CLP Criteria. Therefore, this effect does not indicate significant toxicity in relation to STOT RE classification in GHS. Since no adverse effects have been identified at 50 mg/kg bw/day or below this dose level for either sex, the NOAEL for systemic toxicity is set at 50 mg/kg bw/day.