Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a key, K1 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats at dose levels up to 100 mg/kg bodyweight/day (OECD guideline 422; Edwards, 2018). The NOAEL was considered to be 100 mg/kg bw/day for reproductive toxicity.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-15 - final report date
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
other: (EC) No 440/2008
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Jeffcat LE 30; 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- Physical state/appearance: clear colorless liquid
- CAS number: 65286-55-7
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 30 December 2018
- Purity test date: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature and humidity in darkness
- Stability under test conditions: used/formulated in light
- Solubility and stability of the test substance in the solvent/vehicle: stable in distilled water for at least 15 days when stored refrigerated

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 and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 11 weeks
Females: approx. 12 weeks
- Weight at study initiation:
Males: 270g - 335g
Females: 196g - 240g
- Fasting period before study: not specified
- 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 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: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

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

IN-LIFE DATES: From: 2017-09-12 To: 2017-11-15
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
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 15 days when stored refrigerated. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 4, 12, 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/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
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 stability and homogeneity of the test item formulations were determined at the test facility and results showed that the formulations to be stable for at least 15 days when stored refrigerated.
- Samples of test item formulations were taken on three occasions and analyzed for concentration and the results indicate that the prepared formulations were within 98-104% of the nominal concentration.
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 for females)
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
48 males and 48 females in total; 12 males and 12 females per dose group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat (Study No.:QS12HY)
- 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 working week (except for females during parturition where applicable).
- Parameters: signs of toxicity, ill-health and 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 : 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: Yes
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: Yes
- Time schedule for examinations: 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 44 for males and Day 13 post partum for females)
- Anaesthetic used for blood collection: not specified. Blood samples were obtained from the lateral tail vein.
- Animals fasted: no
- How many 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) 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 44 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 (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 OBSERVATION
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIORAL ASSESSMENT:
- Detailed individual clinical observations were performed for each animal using a purpose built arena.
- The parametes observed were : Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

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.
- Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

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. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
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), Sex of offspring on Day 1, 4 , 7 and 13 postpartum, Clinical condition of offspring from birth to Day 13 postpartum, Individual offspring weights on Day 1, 4, 7 and 13 postpartum (litter weights were calculated retrospectively from this data).

PHYSICAL DEVELOPMENT:
All live offspring were assessed for ano-genital distance on day 1 post partum. Visible nipple count was performed for all male offspring at Day 13 postpartum

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.

GROSS NECROPSY
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHT
- For five males and five females selected from each test and control group.
- The following organs were examined: 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.
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:
Homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup 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 KruskalWallis 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:
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 pregnant females/number of animals mated) x 100
Gestation length: calculated as the number of days of gestation including 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 sites – 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 dosage of 20, 60 and 100 mg/kg bw/day.
At 100 mg/kg bw/day, five males and seven females, respectively, exhibited sporadic instances of noisy respiration between Days 8 and 52 of treatment. Four females at 60 mg/kg bw/day exhibited noisy respiration between Days 8 and 45. At this incidence this observation is likely to be associated with difficulty during the dosing procedure for these particular animals, rather than any underlying effect of systemic toxicity.
One 100 mg/kg bw/day male exhibited pilo-erection and hunched posture on Day 24 of treatment.
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):
Males treated with 60 or 100 mg/kg bw/day had statistically significantly lower body weight gains (p<0.01 - p<0.05) throughout the treatment period in a dose related manner. This resulted in overall body weight gains which were 40% and 50% lower than controls for males treated with 60 or 100 mg/kg bw/day, respectively. Body weight gains for males treated at 20 mg/kg bw/day remained comparable to controls throughout the study.

Females at 100 mg/kg bw/day showed a mean body weight loss during the first week of treatment, and a reduced body weight gain during the second week. This resulted in overall body weight gains during the pre-pairing phase being 97% lower when compared to controls. At 60 mg/kg bw/day, females showed a statistically significant (p<0.05) group mean body weight loss during the second week of treatment, this resulted in overall body weight gains to be 15% lower compared to controls. There was no effect of treatment in females treated with 20 mg/kg bw/day, a statistically significantly higher (p<0.05) body weight gain was noted during the first week of treatment, however, an increase in body weight gain is not considered to be an adverse effect of treatment, and is therefore of no toxicological significance.

During the gestation phase, females treated at 100 mg/kg bw/day showed body weights on Day 14 of gestation which were statistically significantly lower (p<0.05) compared to controls, however, the majority of individual values were within the background control ranges. Females treated with 60 or 100 mg/kg bw/day showed lower body weight gains in relation to controls during the first week of gestation. These females attained statistical significance on Days 0 to 7 (p<0.05 and p<0.001, respectively). Signs of recovery were noted during the second and third weeks of gestation in animals treated with 100 mg/kg bw/day; however, body weight gains remained slightly lower than control but were not as marked. These reductions in body weight gains at 100 mg/kg bw/day were reflected in lower cumulative body weight gains between Days 0 to 14 (p<0.001) and Days 0 to 20 (p<0.05) compared to controls. In contrast, females treated with 60 mg/kg bw/day showed good signs of recovery after the first week of gestation, as body weight gains were comparable to controls.

Females treated with 60 or 100 mg/kg bw/day showed body weights on Day 14 of lactation which were statistically significantly lower (p<0.01) compared to controls. Females treated with 100 mg/kg bw/day showed reductions in body weight gains throughout lactation which achieved statistical significance (p<0.01) between Days 4 to 7 and lower cumulative gains (p<0.01) between Days 1 to 14. Females treated with 60 mg/kg bw/day exhibited comparable body weight gains to control from Days 1 to 4 of lactation, however, reductions in body weight gains were subsequently noted, which achieved statistical significance (p<0.01) from Days 4 to 7.

Body weights and body weight gains were unaffected by treatment at 20 mg/kg bw/day during the gestation and lactation phases.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Male food consumption at 60 or 100 mg/kg bw/day was generally lower in relation to controls from Week 2 of treatment onwards. No such effects were detected for males treated with 20 mg/kg bw/day.

For females, there were no adverse effects noted on food consumption during the pre-pairing phase.

However, throughout gestation statistically significantly (p<0.01 – p<0.001) lower food consumption was apparent throughout this phase. This trend continued during lactation in females treated with 100 mg/kg bw/day as lower food consumption was observed which achieved statistical significance (p<0.01) from Day 4 to 14. In contrast, females treated with 60 mg/kg bw/day showed signs of recovery during lactation as food consumptions were only slightly lower during this phase of the study when compared to controls. No effect on food consumption was detected for 20 mg/kg bw/day females during gestation or lactation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The food conversion efficiency showed a similar trend to the male body weight changes and food intake, as males treated with 60 or 100 mg/kg bw/day values were generally lower than controls. No such effects were detected for males treated with 20 mg/kg bw/day.

For females, the food conversion efficiency showed a similar trend to the female body weight changes, as females treated with 60 or 100 mg/kg bw/day were generally lower than controls.
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, treatment-related
Description (incidence and severity):
At 100 mg/kg bw/day males had a statistically significant increase (p<0.01) in neutrophils in relation to controls, without a dose related response. All individual values from these males exceeded the background control ranges. Females treated with 60 and 100 mg/kg bw/day showed a statistically significant increase in neutrophil count (p<0.05 and p<0.01, respectively) compared to controls in a dose related response. These females also showed a statistically significant increase in lymphocyte counts (p<0.05) in a dose related response. The increased levels of neutrophil and lymphocyte counts for these females, therefore, contributed to the statistically significant increase in the total leukocyte count (p<0.05 and p<0.01, respectively) in a dose related manner. The majority of individual values were within the background control ranges, with isolated individuals exceeding these ranges. The increased values in these parameters may be a response to the extensive vacuolation/vacuolated macrophages of numerous tissues observed at histopathological examination.

Males treated with 100 mg/kg bw/day showed a statistically significant decrease (p<0.05) in hematocrit, however all individual values for these males were within the background control ranges. These males also showed a statistically significant increase (p<0.05) in platelet counts. 3/5 individual male values at 100 mg/kg bw/day exceeded the background control ranges. Due to the effects noted at histopathological examination an association with treatment cannot be discounted.

Treated males and females showed a dose related increase in reticulocyte count, attaining statistical significance for males treated with 60 and 100 mg/kg bw/day (p<0.01) and females at 100 mg/kg bw/day (p<0.05). 4/5 and 5/5 individual male values at 60 and 100 mg/kg bw/day respectively, and 2/5 individual female values at 100 mg/kg bw/day exceeded the background control ranges. Although there is no evidence of red blood cell destruction or anemia which would trigger increased reticulocyte production, and no corresponding histopathological changes in the bone marrow, a relationship to treatment cannot be excluded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Both 60 and 100 mg/kg bw/day males showed a decrease (p<0.05) in alkaline phosphatase (AP) in relation to the controls. All individual values were within the background control range. At 100 mg/kg bw/day, males also showed an increase (p<0.01) in aspartate aminotransferase (ASAT), however 3/5 individual values exceeded the background control range; including one value which was extremely high, and was likely to be the reason for this statistical significance. Due to the histopathological correlates to the liver an associated response to treatment cannot be ruled out.
Females treated with 60 and 100 mg/kg bw/day also showed a statistically significant reduction in alanine aminotransferase (ALAT) in relation to controls. Due to histopathological correlates to the liver an associated response to treatment cannot be ruled out.
At 100 mg/kg bw/day the males showed statistically significant reductions in glucose (p<0.05), albumin (p<0.01), total protein (p<0.01) and bilirubin (p<0.05) compared to controls. These males, as well as all female treatment groups, showed a statistically significant increase (p<0.05) in sodium concentration compared to controls. However, none of these parameters showed a dose related response, and all individual values were within background control ranges. These findings are likely to be the result of normal biological variation and of no toxicological significance.
An increase (p<0.05) in bile acids was noted for females treated with 100 mg/kg bw/day, without a dose relationship when compared to controls. All individual values were within the background control ranges, with 1/5 of the values at the upper end of this range may have influenced the significance of this parameter. This finding is likely to be the result of normal biological variation, however, due to histopathological changes in the liver a relationship to treatment cannot be discounted.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral assessment: There were isolated incidences of noisy respiration from one male each from 60 and 100 mg/kg bw/day dose groups on Day 14 and three 100 mg/kg bw/day males on Day 42 of treatment. Also noisy respiration was noted for three females treated with 100 mg/kg bw/day on four separate occasions. Due to the sporadic nature of these observations this finding was considered to be of no toxicological significance.

Functional performance: There were no changes in functional performance considered to be related to treatment at 20, 60 and 100 mg/kg bw/day.
Males treated with 20 mg/kg bw/day showed a statistically significant increase (p<0.05) in hind limb grip strength. The intergroup difference was confined to one out of the three tests, in the absence of any similar effects at higher dosages, this finding was considered to be incidental and of no toxicological importance.
The final 20% of activity was reduced in males treated with 60 or 100 mg/kg bw/day which attained statistical significance (p<0.05), a dose relationship was not apparent. Overall activity was statistically significantly reduced (p<0.05 - p<0.01) in these animals when compared to controls in a dose related manner. In the absence of any clinical signs of neurotoxicity the intergroup difference was considered to be of no toxicological significance.

Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 20, 60 and 100 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were extensive and unusual changes observed within this study, which were considered to be a direct effect of treatment with the test item.

Vacuolation/vacuolated macrophages were observed in numerous tissues from males treated with 20 mg/kg bw/day and above. The degeneration of hepatocytes in the liver was seen in males of all dose levels and females treated with 60 or 100 mg/kg bw/day. As vacuolation is a part of the degeneration process, this finding was considered to be adverse.

Reduced hematopoiesis in the spleen was present in all males and females treated with the test sustance. The significance of this finding is unclear but unlikely to be significant. A further male treated with the test substance at 100 mg/kg bw/day had cellular depletion.

Atrophy of the thymus was present in two males and one female treated with 100 mg/kg bw/day.

Adrenal Glands: Vacuolation of the cells in the cortex was present in two males and females treated with 20, 60 and 100 mg/kg bw/day.
Aorta: Vacuolation in the wall of the aorta was present in two females given 20 mg/kg bw/day and all males and females given 60 or 100 mg/kg bw/day.
Brain: Vacuolation/vacuolated macrophages of the choroid plexus was present in 4/5 males and all females treated with 100 mg/kg bw/day. It was present in 4/5 males and one female treated with 60 mg/kg bw/day. It occurred in all brain regions where choroid plexus was present but was recorded under cerebrum for clarity.
Eyes: Vacuolation in the ciliary body was present in all females and 2/5 males treated with 100 mg/kg bw/day. It was also present in one female treated with 20 mg/kg bw/day.
Esophagus: Vacuolation in the muscle layer was present in all males and 3/5 females treated with 100 mg/kg bw/day, along with one female treated with either 20 or 60 mg/kg bw/day.
Heart: Vacuolation/vacuolated macrophages occurred in the cardiac muscle cells and/or the wall of the vessels of all males and females treated with 60 or 100 mg/kg bw/day. It was present in one male treated with 20 mg/kg bw/day.
Kidneys: Vacuolation/vacuolated macrophages in the glomeruli was present in 4/5 males and all females treated with 100 mg/kg bw/day. It was present in 3/5 males and females treated with 60 mg/kg bw/day.
Liver: Centrilobular, vacuolation and degeneration was present in all males and females treated with 60 or 100 mg/kg bw/day. It was present in 2/5 males treated with 20 mg/kg bw/day. Vacuolation alone, multifocal and minimal was present in the other three males treated with 20 mg/kg bw/day.
Pancreas: Vacuolation was present in all males and females treated with 100 mg/kg bw/day along with all males and 2/5 females treated with 60 mg/kg bw/day.
Respiratory Tract: Vacuolation was present in the tracheal epithelium and/or the epithelium lining the bronchi of all males and females treated with either 60 or 100 mg/kg bw/day. Vacuolation in the muscle surrounding the bronchi/bronchioles was present in all males and females treated with 100 mg/kg bw/day, most males and females treated with 60 mg/kg bw/day and 3/5 males and females treated with 20 mg/kg bw/day.
Parathyroid Glands: Vacuolation was present in all males and females treated with either 60 or 100 mg/kg bw/day, in which the parathyroid glands were present.
Pituitary Gland: Vacuolation of the posterior lobe was present in seven males and eight females treated with 100 mg/kg bw/day. It was also present in one male treated with 20 mg/kg bw/day and two males and four females treated with 60 mg/kg bw/day.
Prostate: Vacuolation in the muscle tissue within the prostate was present in 8/12 animals treated with 100 mg/kg bw/day and 6/7 treated with 60 mg/kg bw/day.
Seminal Vesicles: Vacuolation in the muscle tissue within the seminal vesicles was present in 11/12 animals treated with 100 mg/kg bw/day and all males treated with 60 mg/kg bw/day which were examined.
Spleen: Vacuolated macrophages were present in all males and 4/5 females treated with 100 mg/kg bw/day and all males and females treated with 60 mg/kg bw/day.
Reduced hematopoiesis was present in all males and females treated with Jeffcat LE 30. A further male treated with 100 mg/kg bw/day had cellular depletion.
Stomach: Vacuolation in the glandular region of the stomach mucosa was present in all males and females treated with 100 mg/kg bw/day and most treated with 60 m/kg bw/day. Vacuolation in the muscularis of the stomach was present in 4/5 males and all females treated with 100 mg/kg bw/day, all males and females treated with 60 mg/kg bw/day along with most males and females treated with 20 mg/kg bw/day. Vacuolar degeneration in the muscularis was present in the remaining male treated with 100 mg/kg bw/day.
Intestines: Vacuolation in the muscularis was present in one or more areas of the intestine in all males and females treated with either 60 or 100 mg/kg bw/day.
Thymus: Atrophy was present in two males and one female treated with 100 mg/kg bw/day. Macrophage vacuolation was present in one further female treated with 100 mg/kg bw/day.
Thyroid Glands: Vacuolation was present in 10/12 males and 8/12 females treated with 100 mg/kg bw/day.
Urinary Bladder: Vacuolation in the muscle of the bladder wall was present in all males and females treated with 100 mg/kg bw/day along with all males treated with 60 and one male treated with 20 mg/kg bw/day.
Uterus: Vacuolated macrophages were present in the walls of the uterus in most animals treated with either 60 or 100 mg/kg bw/day.
Vacuolation of the cells of the uterine wall was present in most animals treated with either 60 or 100 mg/kg bw/day.

Non Productive Matings:
There were a number of non-productive matings within the study, generally spread through the Groups, Control - Female 13 (Male partner 1), Low – Female 42 (Male partner 30) and Female 43 (Male partner 31), Intermediate – Female 63 (Male partner 51) and Female 65 (Male partner 53), High – Female 85 (Male partner 73) and Female 89 (Male partner 77). No findings were noted in the tissues examined, of the animals involved, which could account unequivocally for the lack of pregnancy. The uterus of 2 animals treated with the test substance at 100 mg/kg bw/day showed mild atrophy and one had anestrus morphology in the vagina and the changes in the uterus caused by the test item cannot be discounted.
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle). The uterus of 2 animals treated with the test substance at 100 mg/kg bw/day showed mild atrophy and one had anestrus morphology in the vagina. The follicles and corpora lutea in the ovaries indicated no test item related microscopic findings.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
An evaluation of Thyroxine (T4) in adults show a dose related reduction attaining statistical significance (p<0.001) for adult males treated with 60 and 100 mg/kg bw/day. This could be an associated effect due to the histopathological changes to the thyroid gland.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with all females showing regular cycles during the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating: Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 20, 60 or 100 mg/kg bw/day. The majority of animals mated within four days of pairing, with the exception of one control and one 100 mg/kg bw/day pairing, which both mated after thirteen days of pairing.

Fertility: 1, 2, 2 and 2 females from control, 20, 60 and 100 mg/kg bw/day dose groups were nonpregnant. For the pairings which did not result in pregnancy, no findings were noted in the tissues examined which could account unequivocally for the lack of pregnancy. The uterus of two animals treated at 100 mg/kg bw/day showed mild atrophy and one had anestrus morphology in the vagina and the changes in the uterus caused by the test item cannot be discounted.

Gestation length: The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 20, 60 or 100 mg/kg bw/day.

Litter response: In total 11, 10, 8 and 8 females from control, 20, 60 and 100 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 13 of age. 1, 2, 2 and 2 females from control, 20, 60 and 100 mg/kg bw/day dose groups were non-pregnant. Two females treated at 60 mg/kg bw/day and two females treated 100 mg/kg bw/day had total litter loss post partum. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
100 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
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
100 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
Clinical signs:
no effects observed
Description (incidence and severity):
The 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 20, 60 and 100 mg/kg bw/day.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was considered to be no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 20, 60 and 100 mg/kg bw/day.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring weights of both sexes with maternal treatment of 60 and 100 mg/kg bw/day were initially comparable to controls. However, body weight gains were lower throughout, which achieved statistical significance from Day 4 until termination (p<0.01 - p<0.001). Cumulative body weight gains were also statistically significantly lower for both dosages between Days 1-7 and 1-13. This resulted in lower overall body weights from Day 4 which subsequently achieved statistical significance on Days 7 and 13 of age for both sexes (males: p<0.01 or p<0.001, females: p<0.05 - p<0.001). This finding was more pronounced with maternal treatment of 100 mg/kg bw/day. Litter weights were lower at 100 mg/kg bw/day throughout the lactation phase, and attained statistical significance from Day 4 (p<0.05 p<0.01). Litter weights at 60 mg/kg bw/day were initially comparable to control, however, lower litter weights were subsequently apparent from Day 4 until termination but these failed to achieve statistical significance.

The offspring of females treated with 20 mg/kg bw/day had litter weights that were comparable to or exceeded controls throughout lactation. Offspring body weights and gains were also generally similar to controls, male offspring exhibited statistically significantly lower body weights (p<0.05) between Days 4(AC)-7. However, as male offspring overall body weight gains and actual body weights were comparable to control, this is unlikely to be a true effect of treatment. This is supported by the fact that all individual litter values for male offspring body weights gains for this period were within the background control range.
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
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item on offspring development, as indicated by ano-genital distance on Day 1 post partum at 20, 60, or 100 mg/kg bw/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item on offspring development, as indicated by visible nipple count on Day 13 post partum at 20, 60, or 100 mg/kg bw/day.
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 20, 60 or 100 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
An evaluation of Thyroxine (T4) in offspring (Day 13 of age) was performed; this did not identify any treatment-related findings in the offspring.

There was no effect of treatment with the test item on offspring development, as indicated by ano-genital distance on Day 1 post partum and visible nipple count in male offspring on Day 13 post partum at 20, 60 or 100 mg/kg bw/day.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
There was considered to be no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 20, 60 and 100 mg/kg bw/day.
At 100 mg/kg bw/day, the mean number of implantations was lower than control although this value failed to attain statistical significance. The number of implantation was not particularly low at this dosage and this difference appears to be incidental and unrelated to maternal treatment. The mean total number of offspring born was slightly lower than control but consistent with the previous slightly lower mean number of implantations at this dosage. Liter size on Day 1, 4, 7 and 13 post partum were also slightly lower for females treated with 100 mg/kg bw/day when compared to control litters which again failed to achieve statistical significance.
Live birth index and offspring viability in treated females was comparable to controls.
Sex ratio across all treatment groups was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 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

Discussion of the results

The administration of the test substance to rats at 20, 60 and 100 mg/kg bw/day resulted in statistically significant reductions in body weight development throughout the treatment period for both males and females treated with 60 or 100 mg/kg bw/day compared to controls. A reduction in food consumption for these animals was also apparent throughout the treatment period and with no visible effects on water intake. No such effects were detected in animals of either sex treated with 20 mg/kg bw/day. There were no clinical observations of true systemic toxicity and only sporadic instances of noisy respiration from isolated individuals.  

Statistically significant differences in males and females treated with 60 or 100 mg/kg bw/day were noted in comparison to controls for the hematological and blood chemical parameters measured. Due to the effects apparent at histological examination an association with treatment cannot be discounted for the majority of these differences.  

The evaluation of Thyroxine (T4) in adults and male/female offspring (Day 13 of age) did not identify any treatment-related findings in the offspring. However, the adults did show a dose related reduction attaining statistical significance for adult males treated with 60 and 100 mg/kg bw/day. This could be an associated effect due to the histopathological changes to the thyroid gland.

Mating performance and fertility were unaffected by treatment. However, offspring from 60 or 100 mg/kg bw/day litters showed a reduction (p<0.05 - p<0.001) in body weight, body weight gain and cumulative gains which resulted in statistically significantly lower litter weights at 100 mg/kg bw/day (p<0.05 - p<0.01).  No such effects were noted for offspring from 20 mg/kg bw/day litters.  

Microscopic examination of tissues revealed extensive and unusual changes within this study which were considered to be a direct effect of treatment with the test item. Vacuolation/vacuolated macrophages were present in numerous tissues. Males treated with 20 mg/kg bw/day and females treated with 60 and 100 mg/kg bw/day also had vacuolation/vacuolated macrophages present in the choroid plexus. Vacuolation is seen as part of a degeneration process, degeneration of hepatocytes was seen in the liver of males at all dose levels and females at 60 or 100 mg/kg bw/day.  Although the exact cause of the vacuolation cannot be determined, it may be related to the uptake and/or clearance of the test item or its metabolite(s) or due to phospholipidosis although this is not clear.   It may also be related to a change in, or disruption of, the storage process within the cells. Special staining with Oil-Red-O and PAS did not give any indication of the identity of the contents of the vacuoles.

There were also reduced amounts of splenic hematopoiesis present in all groups treated with the test item. There were no notable correlating changes were apparent in the hematology parameters therefore the significance is unclear but unlikely to be significant.

Based on these findings, a No Observed Adverse Effect Level (NOAEL) for systemic toxicity can be established for females only at 20 mg/kg bw/day within the confines of this study, due to the absence of vacuolation or any further changes in the tissues. This could not be established for males. A No Observed Effect Level (NOEL) for reproductive toxicity was considered to be at least 20 mg/kg bw/day.

Conclusions:
The oral administration of the test substance to rats by gavage at dose levels of 20, 60 and 100 mg/ kg bw/day resulted in treatment related, non-adverse effects. The primary observation in the study was vacuolisation/vacuolated macrophages in numerous tissues from males treated with 20 mg/kg bw/day and above and degeneration of hepatocytes in the liver for males of all dose levels and females treated with 60 or 100 mg/kg bw/day. With regards to vacuolisation/vacuolated macrophages in numerous tissues, the vacuoles observed are considered transient and non-adverse based on scientific peer-reviewed literature regarding weakly basic aliphatic amines (which can be primary, secondary or tertiary amines) and supporting evidence from similar Huntsman compounds within the same chemical family of aliphatic amines. With regards to degeneration of hepatocytes in the liver for males and females, there are no other parameters within the study that supports this observation as being adverse or progressive (i.e., clinical pathology or hematology/clinical blood chemistry). Thus, with no obvious adverse effects at 100 mg/kg bw/day, a NOAEL of 100 mg/kg bw/day is considered acceptable (Lewis et al. 2012).
The reproductive/developmental NOAEL based on various parameters for the test substance from the OECD 422 study (OECD 422 - Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test), dosed at 20, 60 and 100 mg/kg/day, should be set at 100 mg/kg/day.
The primary reproductive/developmental observation in the study was a decrease in fetal body weights (male, female and total litter weight) for the various dose groups compared to the Control Group. However, there was always a positive weight gain throughout the study in each dose group at each body weight measurement timepoint. This observation is considered to be a secondary effect due to lower maternal (female) food consumption in the dose groups compared to Control Group, which is also reflected in maternal (female) body weights. Even though food consumption tended to be lower for dose group animals compared to the Control Group, there was always a positive increase in food consumption throughout the study for all groups.
Thus, with no obvious adverse effects at 100 mg/kg/day (i.e., mortalities) and positive maternal and fetal weight gains, as well as, positive maternal food consumption throughout the study, a NOAEL of 100 mg/kg/day is considered acceptable (Lewis at al, 2012).
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A key, K1 reliable combined repeated dose toxicity study with reproductive/developmental screening was performed according to OECD guideline 422 (Edwards, 2018). The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™: RccHan™:WIST strain rats, for approximately eleven weeks (males) and twelve weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 20, 60 and 100 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (distilled water) over the same period. Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only).

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

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

Vaginal smears were also performed in the morning on the day of termination for all treated females. Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post-partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

There was no mortality observed during the study. There were no clinical signs apparent that were considered to be related to systemic toxicity of the test item.

There were no treatment-related changes in the behavioral parameters in animals of either sex treated with 20, 60 and 100 mg/kg bw/day. Some isolated incidences of noisy respiration from one male each from 60 and 100 mg/kg bw/day dose groups, three males and three females at 100 mg/kg bw/day were noted. However, these observations this finding was considered to be of no toxicological significance due to the sporadic nature.

There were considered to be no treatment related changes in functional performance considered to be related to treatment at 20, 60 and 100 mg/kg bw/day. Males treated with 20 mg/kg bw/day showed a statistically significant increase (p<0.05) in hind limb grip strength but in the absence of any similar effects at higher dosages, this finding was considered to be incidental and of no toxicological importance.

In terms of body weight, the males treated with 60 or 100 mg/kg bw/day had statistically significantly lower body weight gains (p<0.01 - p<0.05) throughout the treatment period in a dose related manner. No differences, compared to control, were observed in males treated at 20 mg/kg bw/day throughout the study.

Females at 100 mg/kg bw/day showed a mean body weight loss during the first week of treatment, and a reduced body weight gain during the second week. At 60 mg/kg bw/day, females showed a statistically significant (p<0.05) group mean body weight loss during the second week of treatment. There was no effect of treatment in females treated with 20 mg/kg bw/day.  

During the gestation phase, females treated with 60 or 100 mg/kg bw/day showed lower body weight gains in relation to controls during the first week of gestation. These females attained statistical significance on Days 0 to 7 (p<0.05 and p<0.001, respectively). Signs of recovery were noted during the second and third weeks of gestation in animals treated with 100 mg/kg bw/day and after the first week of gestation for animals treated with 60 mg/kg bw/day.

Females treated with 60 or 100 mg/kg bw/day showed body weights on Day 14 of lactation which were statistically significantly lower (p<0.01) compared to controls. 

Body weights and body weight gains were unaffected by treatment at 20 mg/kg bw/day during the gestation and lactation phases. Male food consumption at 60 or 100 mg/kg bw/day was generally lower in relation to controls from Week 2 of treatment onwards. The food conversion efficiency showed a similar trend to the male body weight changes and food intake, as males treated with 60 or 100 mg/kg bw/day values were generally lower than controls. No such effects were detected for males treated with 20 mg/kg bw/day. There were no adverse effects noted on food consumption during the pre-pairing phase, however, food conversion efficiency showed a similar trend to the female body weight changes, as females treated with 60 or 100 mg/kg bw/day were generally lower than controls. Throughout gestation statistically significantly (p<0.01 – p<0.001) lower food consumption was apparent. This trend continued during lactation in females treated with 100 mg/kg bw/day but, females treated with 60 mg/kg bw/day showed signs of recovery during lactation. 

No effect on food consumption was detected for 20 mg/kg bw/day females during gestation or lactation.

Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at 20, 60 or 100 mg/kg bw/day.

1, 2, 2 and 2 females from control, 20, 60 and 100 mg/kg bw/day dose groups were nonpregnant. For the pairings which did not result in pregnancy, no findings were noted in the tissues examined which could account unequivocally for the lack of pregnancy. However, the uterus of two animals treated at 100 mg/kg bw/day showed mild atrophy and one had anestrus morphology in the vagina and the changes in the uterus caused by the test item cannot be discounted.

There was considered to be no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 20, 60 and 100 mg/kg bw/day.

Live birth index and offspring viability in treated females was comparable to controls.

There was no effect of treatment with the test item on offspring development, as indicated by ano-genital distance on Day 1 post partum and visible nipple count in male offspring on Day 13 post partum at 20, 60 or 100 mg/kg bw/day.

Offspring weights of both sexes with maternal treatment of 60 and 100 mg/kg bw/day were initially comparable to controls. However, body weight gains were lower throughout which achieved statistical significance from Day 4 until termination (p<0.01 - p<0.001). The offspring of females treated with 20 mg/kg bw/day had litter weights that were comparable to or exceeded controls throughout lactation.

The clinical signs apparent for offspring on the study were typical for the age observed. There were no treatment-related abnormalities detected at necropsy.

An evaluation of Thyroxine (T4) in adults and male/female offspring (Day 13 of age) was performed; this did not identify any treatment-related findings in the offspring. However, the adults did show a dose related reduction attaining statistical significance (p<0.001) for adult males treated with 60 and 100 mg/kg bw/day. This could be an associated effect due to the histopathological changes to the thyroid gland.

Histopathology results revealed extensive and unusual changes which were considered to be a direct effect of treatment with the test item. These changes included Vacuolation/vacuolated macrophages (at the adrenal glands, aorta, brain, eyes, esophagus, heart, kidneys, liver, pancreas, respiratory track, parathyroid glands, Pituitary Gland, prostate, seminal vesicles, spleen, stomach, intestines, Thyroid Glands, Urinary Bladder and uterus), reduced hematopoiesis in the spleen, atrophy of the thymus. As described in the repeated dose section, these effects are considered to be osmotic effects that are transient in nature and non-adverse.

There were a number of non-productive matings within the study, generally spread through the groups. No findings were noted in the tissues examined, of the animals involved, which could account unequivocally for the lack of pregnancy. The uterus of 2 animals treated with the test substance at 100 mg/kg bw/day showed mild atrophy and one had anestrus morphology in the vagina and the changes in the uterus caused by the test item cannot be discounted. The follicles and corpora lutea in the ovaries indicated no test item related microscopic findings.

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).

The oral administration of the test substance to rats by gavage at dose levels of 20, 60 and 100 mg/ kg bw/day resulted in treatment related, non-adverse effects. The primary observation in the study was vacuolisation/ vacuolated macrophages in numerous tissues from males treated with 20 mg/kg bw/day and above and degeneration of hepatocytes in the liver for males of all dose levels and females treated with 60 or 100 mg/kg bw/day. With regards to vacuolisation/vacuolated macrophages in numerous tissues, the vacuoles observed are considered transient and non-adverse based on scientific peer-reviewed literature regarding weakly basic aliphatic amines (which can be primary, secondary or tertiary amines) and supporting evidence from similar compounds within the same chemical family of aliphatic amines. With regards to degeneration of hepatocytes in the liver for males and females, there are no other parameters within the study that supports this observation as being adverse or progressive (i.e., clinical pathology or hematology/clinical blood chemistry). Thus, with no obvious adverse effects at 100 mg/kg bw/day, a NOAEL of 100 mg/kg bw/day is considered acceptable (Lewis et al. 2012).

The reproductive/developmental NOAEL based on various parameters for the test substance from the OECD 422 study (OECD 422 - Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test), dosed at 20, 60 and 100 mg/kg/day, should be set at 100 mg/kg/day. 

The primary reproductive/developmental observation in the study was a decrease in fetal body weights (male, female and total litter weight) for the various dose groups compared to the Control Group. However, there was always a positive weight gain throughout the study in each dose group at each body weight measurement timepoint. This observation is considered to be a secondary effect due to lower maternal (female) food consumption in the dose groups compared to Control Group, which is also reflected in maternal (female) body weights. Even though food consumption tended to be lower for dose group animals compared to the Control Group, there was always a positive increase in food consumption throughout the study for all groups. 

Thus, with no obvious adverse effects at 100 mg/kg/day (i.e., mortalities) and positive maternal and fetal weight gains, as well as, positive maternal food consumption throughout the study, a NOAEL of 100 mg/kg/day is considered acceptable (Lewis at al, 2012).

Annex IX testing:

A testing proposal for a prenatal development study in a first species is included.

Effects on developmental toxicity

Description of key information

In the key, K1 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD guideline 422; Edwards, 2018), an effect on offspring weight was observed but considered to be non-adverse. The NOAEL for developmental toxicity is considered to be 100 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Annex IX testing:

A testing proposal for a prenatal development study in a first species is included.

Justification for classification or non-classification

Based on the results, the test substance is considered not to be classified as reproductive toxicant, according to the criteria laid down in the CLP regulation.