3-butoxypropylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-29 to 2019-11-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

3-butoxypropylamine

EC Number:

240-566-8

EC Name:

3-butoxypropylamine

Cas Number:

16499-88-0

Molecular formula:

C7H17NO

IUPAC Name:

3-butoxypropylamine

Specific details on test material used for the study:

- Name: BOPA, 3-Butoxypropylamine
- CAS number: 16499-88-0
- Physical state/appearance: clear, colorless liquid
- Batch number: PFW160573
- Purity: 99.39%
- Expiry date: 2018-09-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark

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) Ltd., Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 11 weeks old at the start of the treatment
Females: approx. 12 weeks old at the start of the treatment
- Weight at study initiation:
Males: 292-350 g
Females: 193-231g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four 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 and mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.)
- Water (e.g. ad libitum): ad libitum, Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 20 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
- Humidity (%): 50 ± 20
- Air changes (per hours) : at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE: From: 2017-12-19 To: 2018-02-20

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- the test item was prepared at the appropriate concentrations as a solution in arachis oil BP.
- the formulations were shown to be stable for at least 4 hours. Formulations were therefore pepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 0, 7.5, 15, 30/22.5* mg/mL (*dose level and concentration reduced from day 11)
- Amount of vehicle (if gavage): 4 mL/kg of Arachis oil BP. 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: one male: one female basis within each dose group.
- 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 analytical procedure was successfully validated for the test substance in Arachis Oil with reqspect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision.
- The homogeneity and stability of the test substance in Arachis Oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 50 mg/mL.
- Samples of test item formulations were taken on two occasions and analyzed for concentration of the test substance at the test facility.

Duration of treatment / exposure:

- Males: approx. 6 weeks
- Females: up to 8 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.:

30 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose group)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate dose group)

Doses / concentrations 4

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Group 4 (High dose group), up to day 11

Doses / concentrations 5

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

Group 4 (high dose group), from day 11 until the end of the study

No. of animals per sex per dose:

48 males and 49 females in total; 12 males and 12 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (including a 14-day repeated dose oral (gavage) range-finding toxicity study in the rat (Envigo Study Number CM26GG)
- Treatment volume : 4mL/kg body weight of Arachis oil BP.
- 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 wer e then determined to ensure similarity between the treatment groups

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE AND DETAILED CLINICAL 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)
- Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approx. 0830 and as late as possible at weekends and public holidays. The following was recorded for each female: date of pairing, date of mating, date and time of observed start of parturition, date and time of observed completion of parturition
- 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 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 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 p ost 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: Yes
- For males: was calculated retrospectively throughout the study period (with the exception of the mating phase).
- For females during the pre-pairing phase: was calculated throughout retrospectively the study period (with the exception of the mating 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
- Water intake was observed daily by visual inspection of water bottles for any overt changes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (Day 43 for males and Day 13 post partum for females)
- Anaesthetic used for blood collection: No. Blood samples were obtained from the lateral vein.
- Animals fasted: No
- Number of animals per group: 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) 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).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination (Day 43 for males and Day 13 post partum for females)
- Animals fasted: No
- How 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 (A P), 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 observe d 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, prio r 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 approxima tely the same time on each occasion (at least two hours after dosing), under similar laboratory con ditions. 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.
- 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.

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. The stage of the estrous cycle was recorded for each day

Sperm parameters (parental animals):

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle.

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
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.

GROSS NECROPSY
- For all females: the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded
- All adult animals and offspring (including those dying during the study): a full external and internal examination, and any macroscopic abnormalities were recorded
- Examination of offspring: macroscopic external examination and an additional internal examination when macroscopic abnormalities were observed.

ORGAN WEIGHTS
- 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 Vesi cles (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), M ammary 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

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
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, 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:

The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Inter-group variances were 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 was found but the data shows nonhomogeneity of means, the data were 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, pair-wise tests was 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 were also investigated by linear regression. 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).

Reproductive indices:

Pre-coital 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 animals paired ) x 100%
Pregnancy Index (%) : (Number of pregnant females / Number of animals mated) x100%
Gestation length : Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturation 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

he majority of animals of either sex treated with 60 or 120/90 mg/kg bw/day displayed increased salivation for the majority of the treatment period; albeit at a lower extent at the lower dose level. Increased salivation was restricted to one male at 30 mg/kg bw/day on one occasion. Noisy respiration was seen in all animals of both sexes dosed with 120/90 mg/kg bw/day on a number of occasions throughout the study. At 60 mg/kg bw/day noisy respiration was seen on several occasions on four males and five females during the treatment period; in addition only one male and two females at 30 mg/kg bw/day showed the same observation. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered not to represent an adverse effect of treatment.
Incidental observations include one male dosed at 120/90 mg/kg bw/day with staining around the snout on Day 29. One male dosed with 120/90 mg/kg bw/day showed pilo-erection and hunched posture on Day 24; one female dosed with 120/90 mg/kg bw/day showed piloerection on Day 1 after dosing. One female treated with 30 mg/kg bw/day exhibited staining around the eyes on Days 50 and 51. These observations are considered to be incidental and of no toxicological significance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three females dosed with 120 mg/kg bw/day were killed in extremis.
- Female 94 (120 mg/kg bw/day) was killed in extremis on Day 2 due to adverse clinical signs including noisy, labored and gasping respiration, pilo-erection, lethargy, hunched posture and distended abdomen. Necropsy observations included gaseous distention of the cecum, colon, duodenum, ileum, jejunum, rectum and stomach. At histopathology there was marked necrosis in the trachea and death is considered to be related to this change, which possibly reflects esophageal reflux.
- Female 90 (120 mg/kg bw/day) was killed in extremis on Day 5. Previously the animal had shown instances of noisy respiration, and on Day 5 was observed to have noisy and gasping respiration, decreased respiratory rate, pilo-erection, lethargy and hunched posture. Necropsy findings included gaseous distension of the duodenum, ileum, jejunum and stomach. At histopathology there was marked necrosis in the trachea and death is considered to be related to this change, which possibly reflects esophageal reflux.
- Female 91 (120 mg/kg bw/day) was killed on Day 6. The animal displayed similar observations as female 90, with instances of noisy respiration on the day before it was humanely killed, and observations of noisy, labored and gasping respiration, decreased respiratory rate and pilo-erection on Day 6. Necropsy showed gaseous distension in the stomach, and a thin non-glandular region of the stomach, but no notable changes were apparent at histopathology.
- On Day 11, the high dose level was reduced from 120 to 90 mg/kg bw/day.
- One female dosed with 120/90 mg/kg bw/day was found dead during the bleeds procedure on Day 13 post partum (Day 51 of the study). Only occasional observations of noisy respiration and increased salivation were noted prior to the animal being found dead. No notable changes were apparent at necropsy or histopathology and this death was considered to be incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- For males dosed with 120/90 mg/kg bw/day: weight gains were statistically significantly lower (p<0.01 and p<0.05, respectively) than controls for the first two weeks of dosing. Following the reduction of the high dose level from 120 mg/kg bw/day to 90 mg/kg bw/day the subsequent body weight gains for these animals generally became comparable to controls (from Week 3).The overall body weight gain for males treated with 120/90 mg/kg bw/day was 19% lower than controls, which is considered to be due to the initial effects on body weight gains during the first two weeks of treatment.
- For males treated with 30 or 60 mg/kg bw/day: no effects were noted.
- All treated female dose groups: weight gains were generally comparable to controls during maturation, gestation and lactation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- For males dosed at 120/90 mg/kg bw/day: food consumptions for males were slightly lower than controls during the first two weeks of treatment. Improvement was noted for the remainder of the treatment period.
- For males dosed at 30 and 60 mg/kg bw/day: no effect on food consumption throughout the study.
- For females dosed at 120/90 mg/kg bw/day: slightly lower than controls during the first week of dosing (maturation)
- For females dosed at 30 and 60 mg/kg bw/day: food intake were similar to controls during maturation, the gestation and lactation periods.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

- For males dosed at 120/90 mg/kg bw/day: food conversion efficiency was slightly lower than controls during Weeks 1 and 2, which correlate to the body weight changes and food intake during this time.
- For females dosed at 120/90 mg/kg bw/day: no effects were noted.
- For males and females dosed at 30 or 60 mg/kg bw/day: no effects were noted.

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 30, 60 or 120/90 mg/kg bw/day.

- For males dosed at 120/90 mg/kg bw/day: platelet values were statistically significantly lower (p<0.05) than controls
- For males dosed at 30 or 60 mg/kg bw/day: mean corpuscular hemoglobin concentration values were statistically significantly higher (p<0.05) than controls.

For both paramaters above, 4/5 individual values were within normal background control ranges. In isolation, and in the absence of any supporting histopathological changes, these findings are likely to represent normal biological variation. They are considered to be incidental and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 30, 60 or 120/90 mg/kg bw/day.
- Animals (either sex) treated at 120/90 mg/kg bw/day: showed statistical significance (p<0.05) in chloride levels compared to controls (males increased and females reduced). However, all individual values were within background control ranges. Hyperchloremia rarely occurs also and is usually accompanied as a part of parallel shifts in sodium levels. In the absence of any such findings, or any histopathological changes, this increase in males is considered to be a result of normal biological variation and of no toxicological significance. The result for female animals is also considered to be due to normal biological variation and of no toxicological significance.

- Males from all dose groups showed a reduction in alanine aminotransferase (ALAT) levels that attained statistical significance (p<0.05 - p<0.01) without a dose relationship. This enzyme is released into the bloodstream in excess of normal levels as a result of damage affecting the liver, thus a decrease in alanine aminotransferase is not an adverse effect. All values for treated males were within background control normal ranges, whereas 2/5 control values exceeded this range. Therefore, in absence of any histopathological liver findings, this finding considered likely to be due to unusually high control values, and considered to be of no toxicological significance.

- Females treated with 120/90 mg/kg bw/day : showed statistically significantly higher albumin/globulin ratios due to statistically significantly higher (p<0.01) levels of albumin. All values for females treated with 120/90 mg/kg bw/day were within normal historical control ranges, whereas 4/5 control values were within these ranges for both parameters. The remaining control value was significantly lower than the background control range for both albumin and albumin/globulin ratio, and this unusually low value is likely to be the cause of this statistical significance, rather than a true toxicological effect.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

-Behavioral assessments
There were no toxicologically significant effects detected in the behavioral assessments.
Seven males and four females exhibited noisy respiration during the first week of treatment while receiving 120 mg/kg bw/day. A further two females at 120/90 mg/kg bw/day showed noisy respiration during the first and third weeks of the gestation phase. These clinical signs were typical of post dose clinical observations seen and considered to be associated with the irritant nature of the test item rather than systemic or neurological effect.
One female treated with 60 mg/kg bw/day showed pilo-erection of a moderate grade and hunched posture.This isolated finding was considered to be incidental and of no toxicological significance
No such effects were detected in male animals treated with 60 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day
-Functional performance.
There were no toxicologically significant changes in functional performance.
Males treated with 60 and 120/90 mg/kg bw/day showed a statistically significant reduction (p<0.05) in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.
- Sensory reactivity:
There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Premature decedents
Females 94 and 90 (120 mg/kg bw/day): The animals were killed for welfare reasons on Day 2 and 5 respectively after showing gasping/laboured respiration and general malaise. At necropsy the gastrointestinal tract exhibited gaseous distension. At the microscopic examination there was marked necrosis in the trachea and death is considered to be related to this change, which possibly reflects esophageal reflux.

Female 91 (120 mg/kg bw/day) was killed for welfare reasons on Day 6 with similar clinical signs and necropsy changes but with no notable changes were apparent at histopathology.

After reduction of the dose level there were no further deaths until Day 51, when Female 87 (120/90 mg/kg bw/day) died during the bleed procedure. There were no notable changes were apparent at necropsy or histopathology and this death was considered most likely to be incidental and unrelated to treatment.


Histopathological examination of tissues from the control and 120/90 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

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 30, 60 or 120/90 mg/kg bw/day.

Reproductive function / performance (P0)

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 30, 60 or 120/90 mg/kg bw/day, with the majority of females showing regular cycling. At necropsy, the majority of females were in di-estrus, with one control and one 30 mg/kg bw/day animal each in estrus.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no consistent treatment-related pathological findings in the testes following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle)

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 30, 60 or 120/90 mg/kg bw/day.
- Fertility: There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy at 30, 60 or 120/90 mg/kg bw/day. There were two non-productive matings at 30 mg/kg bw/day, both pairs showed no changes in the tissues that could account for the lack of pregnancy.
- Gestation: The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 30, 60 or 120/90 mg/kg bw/day.
- Litter responses: There were two females treated with 30 mg/kg bw/day that were non pregnant and one female dosed with 60 mg/kg bw/day had a total litter loss post partum. The following assessment is generally based on the 12, 10, 11 and 10 females that successfully maintained a litter to Day 13 of age at 0 (Control), 30, 60 and 120/90 mg/kg bw/day respectively. Although one female dosed with 120/90 mg/kg bw/day was found dead at Day 13 post partum, as the female had maintained a litter to offspring termination, it is included in this assessment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at treatment of 30, 60 or 120/90 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no obvious effect of maternal treatment on the survival of the offspring from birth to termination (Day 13 of age) at 30, 60 or 120/90 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on offspring body weights, offspring body weight gains and litter weights for animals dosed with 30, 60 or 120/90 mg/kg bw/day.
Body weight for female offspring with maternal treatment of 30 mg/kg bw/day were found to be statistically significantly lower than controls on Day 4 (both before and after culls), and offspring body weight gain for these offspring was statistically significantly lower between Days 1 to 4 (before cull). In the absence of any similar effects in male offspring at 30 mg/kg bw/day or either sex of offspring at dose levels of 60 and 120/90 this finding is considered to be incidental. This is supported by the fact that all individual mean litter values for female offspring body weight and bodyweight gain at 30 mg/kg bw/day were within historical control normal ranges.

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:

not examined

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 systemic effect of maternal treatment at 30, 60 or 120/90 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
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 30, 60 or 120/90 mg/kg bw/day.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

> 90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical verification

Method validation

The specificity of the analytical method was demonstrated by the absence of a peak at the characterisation retention time for test substance in the control sample chromatogram.

The calibration data for the calibration standards of the test item was found to have a linear correlation within the calibration range of 0.05045 mg/mL to 0.2018 mg/mL. The R2 fit of the calibration curve to the data was 0.9999, and was considered to be acceptable.

Method accuracy (recovery) and precision were confirmed. A mean recovery value of 96% (CV=2.97%, n=5) was obtained for 2.5 mg/mL and 98% (CV=0.678%,n=5) was obtained for 50 mg/mL.

The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and stability of dose formulations

The homogeneity and stability of test substance is Arachis Oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 50 mg/mL. Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%. The homogeneity and stability was confirmed for the test substance in Arachis oil formulations at nominal concentrations of 2.5 and 50 mg/mL when stored at ambient temperature for 4 hours.

Test item formulations

The results indicate that the prepared formulations were within 91 - 102% of the nominal concentration.

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

Administration of the test substance in the rat for fourteen consecutive days resulted in all animals of either sex treated with 200 mg/kg bw/day being found dead on Day 2, after receiving two consecutive daily doses of the test item. No clinical signs were observed prior to death. Macroscopic examination revealed reddened glandular regions of the stomach for all males and one female. At 100 mg/kg bw/ day any effects seen were primarily due to one male animal showing body weight loss and clinical signs between days 1 to 4 of treatment. One additional male exhibited noisy respiration on Day 6 only. No further effects were observed in the remaining one male and three females.

Based on the findings of this study, 200 mg/kg bw/day was deemed too high for continued dosing, due to deaths at this dose level. Therefore, the recommended dose levels for the main study are 30, 60 and 120 mg/kg bw/day respectively.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test administration to rats by gavage at dose levels of 30, 60 and 120 mg/kg bw/day resulted in the premature death of three females within the first week of treatment at 120 mg/kg bw/day. Microscopic examination revealed marked necrosis of the trachea due to possible esophageal reflux in two of these animals. This dose level was reduced to 90 mg/kg bw/day on Day 11 of study and there were no further treatment-related deaths. Based on the findings in this study the No Observed Adverse Effect Level (NOAEL) was considered to be at least 90 mg/kg bw/day for systemic toxicity in both males and females. The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 90 mg/kg bw/day. The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 90 mg/kg bw/day. The substance is considered not to be classified as reproductive toxicant.