Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- oral, screening (OECD 422) on Butylaminoethanol; rat: NOAEC (reproductive and developmental parapmeters; males and females) = 240 mg/kg bw/day; NOAEL (systemic males) = 120 mg/kg bw/day (mid dose tested); NOAEL (systemic females) = 240 mg/kg bw/day (high dose tested)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019 Jan 12 to 2020 Jan 28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on July 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Details on species / strain selection:
This test system for this study was the albino rat. It has been historically shown to be an acceptable model for developmental and reproductive toxicity testing and is recommended by the OECD and other regulatory authorities. The results are of value in predicting the toxicity of the test item to humans.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility (ABF), Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11-13 wks
- Weight at study initiation: At the initiation of treatment, the body weight of rats was within ±20% of the mean body weight for each sex
- Housing: Throughout the experimental period, male and female rats were housed in groups of 2 or 3 rats/sex/cage, except during the mating period where rats were housed in groups of 2 rats/cage (one male plus one female). The mated female rats were thereafter caged individually. Enrichment material (wooden chew block) was provided in all cages. Nesting material was provided near parturition (from gestation day 17). During the study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless-steel top grille having provision for polypropylene water bottle with a stainless steel drinking nozzle. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on 5-tier racks. Chemical and microbial contaminant analyses were conductedat six-month intervals.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Rats were fed ad libitum with standard rodent diet. The quality of food was monitored.
- Water (e.g. ad libitum): Rats were provided reverse osmosis (RO) water ad libitum (from a RO water filtration system) in polypropylene bottles. The quality of water was monitored.
- Acclimation period: A greater number of rats than required for the study were ordered to permit the selection of individuals prior to any treatment. A total of 66 male and 66 female rats were received into the experimental room. The rats were acclimatised for 5 days before initiation of pre-treatment oestrous cycle evaluation. The body weight variation of the rats was within ±20% of the mean body weight at the beginning of acclimatisation. During the acclimatisation period, rats were observed twice daily for mortality and morbidity and once daily for clinical signs.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): February 2019: 20-23, March 2019: 20-24, April 2019: 20-24, May 2019: 20-23 (min-max)
- Humidity (%): February 2019: 65-66, March 2019: 58-67, April 2019: 65-67, May 2019: 65-66 (min-max)
- Air changes (per hr): February 2019: 21, March 2019: 20, April 2019: 22, May 2019: 18
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with vehicle (RO water). The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing. Dose formulations of all test item groups were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on solubility determinations performed at JRF RO water was selected as the vehicle.
- Concentration in vehicle: 0, 60, 120, 240 mg/kg bw/day
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage:1:1
- Length of cohabitation: until mating occured or 2 weeks had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability of the Test Item in the Vehicle: The stability of a.i. concentration in the vehicle was determined prior to initiation of the study using a validated analytical method. Based on the results obtained, the test item in the vehicle was stable for 8 days.
- Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle: The a.i. concentration and homogeneity of dose formulations were analysed by the Department of Chemistry, using a validated analytical method:
Two sets of samples (10 samples per set) were collected by sampling three aliquots (top, middle and bottom layers) from each test concentration accompanied by one aliquot (from the middle layer) sampled from the vehicle control. Sampling for dose formulation analysis was performed before the start of treatment and twice during the treatment period. One set of samples was used for analyses and the other set was stored refrigerated as back up sample. The second set of samples were disposed of during report finalisation. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis was ± 10 % from the nominal value and % CV <10.
Duration of treatment / exposure:
- The dosing of both sexes was initiated 2 weeks prior to mating and continued during the mating period.
- After mating, the male rats were further dosed up to and including the day before scheduled sacrifice (when 73 % of the females had delivered).
- Female rats were dosed during pregnancy and up to post-partum day 14.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day
Remarks:
control
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
240 mg/kg bw/day
No. of animals per sex per dose:
15 rats / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a 14-day dose range finding (DRF) study. In the 14-day DRF study, at high dose i.e., 400 mg/kg bw/day, 3 mortalities were observed with toxicity clinical signs. At the mid and low doses, i.e., 125 and 40 mg/kg bw/day, respectively, no signs of toxicity were observed. On this basis, 60, 120 and 240 mg/kg bw/day, were selected for the main OECD 422 study.
- Fasting period before blood sampling for clinical biochemistry: Rats were deprived of food overnight (but allowed water ad libitum) prior to blood collection.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed once daily during treatment periods for any visible clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of all male rats was recorded on the first day of dosing and at weekly intervals thereafter. The body weight of all female rats was recorded on the first day of dosing and at weekly intervals during the pre-mating period. During the gestation period, female rats were weighed on gestation days 0, 7, 14, and 20. During the lactation period, female rats were weighed within 24 hours of parturition (day '0' post-partum/lactation day), on post-partum days 4, 7, 14 and on the day of terminal sacrifice. Parturition days '0' was defined as the day on which the female littered.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The food consumption was determined by difference between the weight of the feed given to the rats and the feed remaining at the end of the specified interval. Food weights of male rats were determined weekly during the pre-mating and post-mating periods.In female rats during the pre-mating period, food weights were recorded at weekly intervals. During the gestation period, food weights were measured on days 0, 7, 14 and 20. During the lactation period, food weights were measured on days 0, 4, 7 and 14. Additional food was offered as needed and included in the food consumption determinations. Food consumption was not measured during the mating period.

OTHER:
- Neurobehavioral Observation (NBO): To assess the behavioral and neurological status of each rat, the NBO parameters listed below were evaluated prior to initiation of treatment and at weekly intervals thereafter. Home Cage Observation (posture, clonic movement, tonic movement) , Handling Observation (ease of removing animal from cage, handling reactivity, palpebral closure, lacrimation, eye examination, piloerection, skin examination, salivation), Open Field Observations (gait, mobility, arousal, vocalisations, rears, respiration, clonic movement, tonic movement, urination, defecation, sterotypy, bizarre behaviour)
- Functional Observational Battery (FOB): The following FOB and NBO parameters were performed in last week of sacrifice using five randomly selected rats/sex/group towards the end of the study: sensory reactivity measurements (approach response, touch response, click response, pupil response, tail-pinch, response, air righting reflex) hind limb foot splay, grip strength, motor activity.
Oestrous cyclicity (parental animals):
Oestrous cycle length and pattern were evaluated by vaginal smear of individual female rats during the two-weeks pre-treatment period. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear from each pregnant rat was also evaluated on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.
Sperm parameters (parental animals):
Parameters examined in [parental high dose group and control] male parental generations:
- testis weight, epididymis weight and examination of caput, corpus and cauda by evaluation of a longitudinal tissue section and evaluation of leukocyte infiltration, change in prevalence of cell types and phagocytosis of sperm, aberrant cell types and phagocytosis of sperm
- stages of spermatogenesis and the histopathology of interstitial testicular cell structure, spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weights (recorded on PND 0, 4, 7, 14), anogenital distance (AGD), presence of nipples/areolae in male pups (PND 13).
Blood was collected by decapitation for serum thyroid hormone analysis from two surplus pups/litter (when available) on postnatal day 4, and two pups/litter on the day of terminal sacrifice.

GROSS EXAMINATION OF DEAD PUPS:
Yes,. Pups found dead on the day of littering were examined for possible defects and cause of death and discarded in the absence of any gross findings.

Other: Each pup was observed for the presence of milk in the stomach on PND 0 to ensure adequate maternal nursing care.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after 73 % of females had delivered.
- Maternal animals: All surviving animals Female rats were sacrificed on LD 15. Females that did not mate were sacrificed 25 days after the last day of the mating period. Female rats not delivered by gestation day 25, were sacrificed on gestation day 25.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination (weighed and preserved (*)): Testes*, epididymis*, levatorani plus bulbocavernosus muscle complex (LABC)*, Cowper's glands*, glans penis*, ovary*, thyroid gland*, seminal vesicles with coagulation gland*, prostate (dorsolateral and ventral)*, uterus with oviducts and cervix*, gross lesions, vagina, male mammary gland, pituitary, liver*, kidney*, adrenals*, heart*, thymus*, spleen*, brain (cerebrum, cerebellum, and medulla/pons)*, spinal cord (at three levels; cervical, mid-thoracic and lumbar), stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon, rectum), eye, trachea, lungs (preserved by inflation with fixative and then immersion), urinary bladder,lymph nodes (mandibular, mesenteric), sciatic nerve, skeletal muscle and bone, bone marrow (Femur).

CLINICAL PATHOLOGY OBSERVATIONS
Haematology Parameters
Clinical Chemistry Parameters
Urinalysis Parameters

THYROID HORMONE ANALYSIS
Serum thyroid hormones (T4, TSH, and T3) levels were analysed from parental male rats, parental female rats and terminally sacrificed
Postmortem examinations (offspring):
SACRIFICE
- F1 Pups were sacrificied on PND 14. Culled pups on PND 4, which were not subjected for blood collection, were sacrificed. Pups which were found dead on PND 0 were subjected for gross examination. Pups without gross lesions were discarded after examination.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Tissues as spezified above were prepared for microscopic examination (weighed and preserved (*).
- The thyroid gland was excised, collected, weighed and preserved from one male pup and one female pup (wherever available)/litter on the day of terminal sacrifice.

THYROID HORMONE ANALYSIS
- Serum thyroid hormones (T4, TSH, and T3) levels were analysed from terminally sacrificed pups (PND 14). Serum thyroid hormone (T4) level was analysed from PND 4 pups.
Statistics:
Study observations were recorded in standard raw data format. Individual rat data have been provided in the report and all data have been summarised in tabular form. Statistical analysis was carried out using validated software developed at JRF. Non-pregnant rats were excluded from statistical analysis. Assumed pregnant rats were excluded from statistical analysis of gestation phase parameters.

Data such as body weight, body weight gain, food consumption, hind limb foot splay, grip strength, organ weight, organ weight ratio, number of implantations, pre-natal loss, post-natal loss, male sex ratio, estrous cycle length, haematology and clinical chemistry parameters, some urinalysis parameters and litter parame ers (pups body weight and pups body weight gain) were subjected to Shapiro- Wilk's test checking normality wherever applicable, followed by Bartlett's test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunett's t-test. When the data did not meet the normality, data was transferred to log form to check the normality again. When log transfer data did not meet the normality, a Kruskal-Wallis test was performed to calculate significance. When the data did not meet the homogeneity of variance, statistical analysis was extended following a decision tree as provided by Gad, S.C., 2007. AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.

Count data {viz., litter size, number of implants, pre-coital interval, duration of gestation, number of estrous cycles, urination count, defecation count, rearing count, motor activity (total, fine and ambulatory)} were subjected to non-parametric test either the Kruskal-Wallis test or Mann-Whitney test.

Non-parametric data such as gestation index, parturition index, pregnancy rate, survival index, mortality index, live birth index and fertility index were analysed using the Chi-Square test.
Reproductive indices:
Pre-natal loss, post-natal loss, mating index, gestation index, live birth index, parturition index, fertility Index, Ano-genital Distance normalised.
Offspring viability indices:
Pup survival Index
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All rats belonging to the control, low and mid dose groups were normal throughout the study period.
Salivation and snuffles were observed in male and female rats of high dose group. Salivation was considered toxicologically insignificant in nature. Snuffles were considered as test item related effect but non-adverse in nature due to absence of effect in remaining rats and no gross observations.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No treatment related mortality or morbidity was observed during the study period in control and test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
- Statistically significantly lower mean body weight was observed in the high dose group on treatment days 22, 29, 36, 43 and 50 when compared with that of the control group (statistically significant decrease of 9 % from control).
- The mean body weight gain of the low and mid dose groups was comparable with the control group, body weight gain in low dose goup (days 15-22) and mid dose goup (days 1-8) were considered incidental and not treatment related. Statistically significant reductions in mean body weight gain in the high dose group during treatment days 1-8, 15-22, 22-29 and 1-5 were considered treatment related due to continous reduction in body weight gain. Body weights of male rats belonging to high dose group were <10 % lower than control male rats, the body weight gains were approximately 60 % lower than control during the study. Overall food consumption was about 12 % lower than control. Hence, changes observed in high dose group were considered as treatment related adverse in nature.

Females:
- The mean body weight of the low, mid and high dose groups was comparable with the control group.
- The mean body weight gain of the low, mid and high dose groups was comparable with the control group. Incidental reduction (statistically significant) in body weight gain in the high dose group during pre-mating day 1-8 was observed when compared with the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
- The mean food consumption of the low and mid dose groups was comparable with that of the control group.
- A statistically significant reduction in food consumption was observed during treatment days 1-8, 8-15 and overall (days 1-50) in the high dose group when compared with the control group. These observed changes were considered as related to treatment in presence of notable change in body weight and body weight gain.

Female:
- The mean food consumption of the low, mid and high dose groups was comparable with the control group.
- However, some incidental changes were observed, as in the mid and high dose group a statistically significant decrease in mean food consumption during pre-mating day 1-8 was observed when compared with the control group.
- A statistically significant decrease in food consumption was observed during gestation day 0-7 in the low and high dose groups when compared with the control group. Changes observed during pre-mating and gestation period were incidental in nature based on comparable food consumption during rest of treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item treatment did not lead to any treatment related alteration in haematological parameters, beside of the following changes, which were in the end classified as unrelated to test item treatment:
- A statistically significant increase was noted in reticulocyte count in high dose group males. It was considered unrelated to test item treatment in absence of effects in other red blood cell related parameters (RBC, haemoglobin and haematocrit).
- A statistically significant increase was observed in lymphocytes in low dose group males which was not considered as related to test item treatment, due to absence of dose dependency.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item treatment did not lead to any treatment related alteration in clinical chemistry parameters, beside ofthe following:
- In high dose males, statistically significant decreases in total protein, globulin, sodium and chloride were noted. These alterations could be related to test item treatment and were not considered as adverse as values were in historical ranges except total protein in 1 male rat of the high dose group. Treatement related histopathological lesions were not observed in liver and kidneys which indicates that the effects were not adverse.
- Statistically significant increase was noted in urea and BUN in low dose females which was not considered as related to test item treatment, due to absence of dose dependency.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis did not reveal any test item related effect.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
NOB:
- Home Cage Observations: All rats from treatment and control groups revealed normal postures when asleep (curled up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were absent.
- Handling Observations: NBO performed during handling of rats did not reveal any abnormality related to treatment. All rats revealed normal behavior during removal (very easy - animals sit quietly) and handling (easy - alert, limbs put against the body). None of the rats showed lacrimation, salivation or piloerection. Eyelids were wide open in all rats. Eye and skin examination of rats from all groups did not reveal any abnormality.
- Open Field Observations: All rats from treatment and control groups showed normal gait, mobility, arousal and respiration during the two minutes observation period. Clonic and tonic movements, stereotypy and bizarre behavior were absent. No treatment related significant changes were observed in vocalisation, rearing, and urination and defecation counts of male and female rats from treatment groups when compared with the control group. Some incidental changes were noted in male rats, including statistically significant decrease in rearing count at weeks 2, 3 and 4 in the high dose group and defecation count in the low dose group at week 7 when compared with the control group. Some incidental changes were noted in female rats, viz., a statistically significant decrease in rear count at week 3 in the high dose group and urination count at pre-treatment week in the low, mid and high dose group when compared with the control group. The findings were considered as incidental based on the lack of findings in other related parameters (motor activity). During 3 minutes of open field observation, snuffle in male rat No 91 and 94 at week 5, 6, 7, and last week of terminal sacrifice whereas in female rat No 119 at week 5, 6, and 7 was observed.
Snuffles were considered as test item related effect (as it was also observed during daily clinical signs observation, as well) but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung.

FOB:
- The motor activity counts of male and female rats from all treatment groups were comparable with the control group.
- Sensory reactivity parameters viz., approach response, touch response, click response, pupil response, tail pinch response and air righting reflex in all treatment groups were comparable with the control group.
- The hindlimb and forelimb grip strength values of rats from treatment groups were comparable with the control group.
- The hindlimb foot splay values of rats from treatment groups were comparable with the control group.
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):
Treatment with the test item did not cause histopathological alterations in any of the organs, beside of the following:
- The microscopic lesions observed in some of the organs were at lower rate of occurrence in control and high dose group and with minimal to mild severity. Hence, those lesions were spontaneous or incidental in nature representing the normal physiological/metabolic or congenital changes and not treatment related. Moreover, in organs showing statistical alterations in organ weight (heart, adrenals, prostate, testes, seminal vesicles with coagulating gland and thymus), did not reveal any test item related lesions and observed lesions were incidental or spontaneous in nature.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Thyroid Hormone Analysis: Thyroid hormone i.e., T3, T4 and TSH serum levels were comparable in males, females and pups (PND 4 and 14) when compared with that of the control group.
- External examination of rats did not reveal any abnormality.
- Home Cage Observations and Handling Observations: no abnormalities related to treatment.
- Open Field Observations: some incidental changes (statistically significant) decrease in rear count. The findings were considered as incidental based on the lack of findings in other related parameters (motor activity). Snuffles were considered as test item related effect (as it was also observed during daily clinical signs observation, as well) but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung.
- Motor Activity, Sensory Reactivity Measurements, Grip Strength, Hindlimb Foot Splay: no effects observed.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean number of oestrous cycles and mean number of oestrous cycle lengths were comparable between test item groups and the control group.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
240 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: systemic toxicity
Clinical signs:
no effects observed
Description (incidence and severity):
All pups belonging to the control and test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no treatment related pup mortality and mortality index observed in test item treated groups when compared with that of the control group. Except, a statistically significant increase in the mortality index of female pups belonging to high dose group was observed during post-natal day 0-4 when compared with the control group. This was due to the complete litter loss of 1 rat (rat N° 108) out of 11 rats of high dose group dams i.e., changes were limited to one rat (and were not spread across the group), hence the observed changes were considered unrelated to treatment and incidental in nature.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain of males, females and combined males and females were comparable between all test item treated groups and the control group.
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
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The ano-genital distance of male, female and composite of male and female pups was comparable with the control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No male pups belonging to either the control or test item treated groups showed retention of nipples.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Terminal body weight and thyroid gland weights (both absolute and relative) of PND 14 pups (male and female) were comparable with the control group.
Other effects:
no effects observed
Description (incidence and severity):
Litter Size and Male Sex Ratio: No treatment related differences were observed in mean counts of male pups, female pups, and combined male and female pups when compared with the control group except for a statistically significant decrease in mean count of males on post-natal day 0 in high dose group. This change was considered as unrelated to treatment as combined male and female pups counts was comparable with that of the control group. The male/female sex ratio of test item treated groups was comparable to that of the control group.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
240 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
other: Post-natal growth
Reproductive effects observed:
no

Fertility : Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats and mating index were comparable with the control group. The duration of the gestation days and pre-coital interval were comparable among all the test item treated groups with the control group.

Pre and Post-natal Data: The mean number of implants, pre and post-natal loss, mean percentage of pre and post-natal loss were comparable between all test item treated groups and the control group. The percent pre-natal loss was higher in high dose group compared to control group though it lacks statistical significance. This was due to all pups belonging to rat No 118 were found either dead or cannibalised on PND 0.

The percent post-natal loss was higher in high dose group compared to control group though it lacks statistical significance. This was due to the complete litter loss by PND 1, of rat N° 108, belonging to high dose group. These changes were limited to one rat (and were not spread across the group), hence the observed changes were considered unrelated to treatment and incidental in nature

Live birth index of test item treated groups was comparable with the control group.

Survival index of test item treated groups was comparable with that of the control group. A statistically significant decrease in live birth index of males and composite of males and females were observed in high dose group when compared with the control group. A statistically significant decrease in survival index of females was observed during post-natal day 0-4 in high dose group when compared with the control group. These findings were due to complete litter loss of rat N° 108, hence these observed changes were considered as unrelated to treatment.

Conclusions:
- NOAEL for systemic toxicity was 120 mg/kg bw/day for males based on effects on body weight, body weight gain and food consumption in male rats receiving 240 mg/kg bw/day.
- NOAEL for systemic toxicity was 240 mg/kg bw/day for female rats based on no effect on systemic endpoints.
- NOAEL for reproductive and fertility toxicity was 240 mg/kg bw/day based on no effect on fertility and reproductive performance.
- NOAEL for developmental toxicity was 240 mg/kg bw/day based on no effect on mortality, clinical signs and postnatal growth.
Executive summary:

The study was conducted to evaluate the toxic characteristics, systemic toxicity, reproduction/developmental toxicity and neurotoxicity of N-Butylaminoethanol when administered by gavage to male and female Wistar rats during pre-mating, mating, gestation, and lactation periods.

Methods: 15 rats / sex / group were treated with N-Butylaminoethanol receiving the following dose levels: Group No - G1 (control): 0 mg/kg bw/day; G2 (low dose): 60 mg/kg bw/day; G3 (mid dose): 120 mg/kg bw/day; G4 (high dose): 240 mg/kg bw/day. Males were treated two weeks prior to mating during the mating (two weeks) and until approximately 73 % of the females had delivered. Total number of treatment days was 50. Females were treated two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery. The following parameters were analysed: mortality/morbidity, clinical signs, body weight, food consumption, oestrous cycle, neurobehavioural observation (NBO), functional observational battery (FBO), pub observation, blood collection, clinical pathology, thyroid hormone, organ weights, histopathology. Males were sacrificed when ~80 % of females had delivered, females in LD15 and pups on PND 14.

Results: The results of dose formulation analyses were within an acceptable range of ± 10 % of the nominal concentration and the Coefficient of Variation (% CV) was less than 10. Thus, the prepared dose had an acceptable active ingredient (a.i.) concentration and were homogeneously prepared.

Low and Mid dose: No signs of toxicity on clinical signs, mortality, body weight, body weight gain, feed consumption, oestrous cycle, NBO, FOB, clinical pathology, and organ weight (absolute and relative) were observed. No treatment related macroscopic and microscopic lesions were observed.

No treatment related effect on fertility index, mating index, gestation index, percent of pregnant female, duration of the gestation period, number of implants, prenatal and postnatal loss (number and percent) was observed. No effect on pup body weight, body weight gain, litter size and male sex ratio was observed in F1 pups. No effect on endocrine sensitive endpoints (viz., AGD, nipple retention and thyroid weight and hormone level).

High dose: No sign of toxicity on mortality, NBO, FOB, oestrous cycle, organ weight, clinical pathology (absolute and relative) was observed. No treatment related macroscopic and microscopic

lesions were observed.

Salivation is toxicologically insignificant, hence considered as non-adverse in nature. Snuffles were considered as test item related effect but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung. In male rats, treatment related effects were observed on body weight (< 10 %), terminal body weight (< 9 %), overall body weight gain (~< 60 %), and overall food consumption (< 12 %). These endpoints were consistently decreased during treatment period. Hence, changes were considered as treatment related adverse in nature.

No treatment related effect on fertility index, mating index, gestation index, percent of pregnant females, duration of the gestation period, number of implants, prenatal and postnatal loss (number and percent) was observed. No effect on pup body weight, body weight gain, litter size and male sex ratio was observed in F1 pups. No effect on endocrine sensitive endpoints (viz., AGD, nipple retention, thyroid weight and hormone level).

Conclusion:

Based on results of this study, it was concluded that:

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 120 mg/kg bw/day for males based on effects on body weight, body weight gain and food consumption in male rats receiving 240 mg/kg bw/day.

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 240 mg/kg bw/day for female rats based on no effect on systemic endpoints.

- NOAEL for reproductive and fertility toxicity was 240 mg/kg bw/day based on no effect on fertility and reproductive performance.

- The NOAEL for developmental toxicity was 240 mg/kg bw/day based on no effect on mortality, clinical signs and postnatal growth.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
240 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

There is a reproductive dose toxicity study available for butylethanolamine.

An OECD 422 screening study was conducted to evaluate the toxic characteristics, systemic toxicity, reproduction/developmental toxicity and neurotoxicity of N-Butylaminoethanol when administered by gavage to male and female Wistar rats during pre-mating, mating, gestation, and lactation periods. 15 rats / sex / group were treated with N-Butylaminoethanol receiving the following dose levels: Group No - G1 (control): 0 mg/kg bw/day; G2 (low dose): 60 mg/kg bw/day; G3 (mid dose): 120 mg/kg bw/day; G4 (high dose): 240 mg/kg bw/day. Males were treated two weeks prior to mating during the mating (two weeks) and until approximately 73 % of the females had delivered. Total number of treatment days was 50. Females were treated two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery. The following parameters were analysed: mortality/morbidity, clinical signs, body weight, food consumption, oestrous cycle, neurobehavioural observation (NBO), functional observational battery (FBO), pub observation, blood collection, clinical pathology, thyroid hormone, organ weights, histopathology. Males were sacrificed when ~80 % of females had delivered, females in LD15 and pups on PND 14.

The results of dose formulation analyses were within an acceptable range of ± 10 % of the nominal concentration and the Coefficient of Variation (% CV) was less than 10. Thus, the prepared dose had an acceptable active ingredient (a.i.) concentration and were homogeneously prepared.

In the Low and Mid dose group animals, no signs of toxicity on clinical signs, mortality, body weight, body weight gain, feed consumption, oestrous cycle, NBO, FOB, clinical pathology, and organ weight (absolute and relative) were observed. No treatment related macroscopic and microscopic lesions were observed.

No treatment related effect on fertility index, mating index, gestation index, percent of pregnant female, duration of the gestation period, number of implants, prenatal and postnatal loss (number and percent) was observed. No effect on pup body weight, body weight gain, litter size and male sex ratio was observed in F1 pups. No effect on endocrine sensitive endpoints (viz., AGD, nipple retention and thyroid weight and hormone level).

In the High dose group animals, no signs of toxicity on mortality, NBO, FOB, oestrous cycle, organ weight, clinical pathology (absolute and relative) was observed. No treatment related macroscopic and microscopic lesions were observed.

Salivation is toxicologically insignificant, hence considered as non-adverse in nature. Snuffles were considered as test item related effect but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung. In male rats, treatment related effects were observed on body weight (< 10 %), terminal body weight (< 9 %), overall body weight gain (~< 60 %), and overall food consumption (< 12 %). These endpoints were consistently decreased during treatment period. Hence, changes were considered as treatment related adverse in nature.

No treatment related effect on fertility index, mating index, gestation index, percent of pregnant females, duration of the gestation period, number of implants, prenatal and postnatal loss (number and percent) was observed. No effect on pup body weight, body weight gain, litter size and male sex ratio was observed in F1 pups. No effect on endocrine sensitive endpoints (viz., AGD, nipple retention, thyroid weight and hormone level).

Conclusion:

Based on results of this study, it was concluded that:

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 120 mg/kg bw/day for males based on effects on body weight, body weight gain and food consumption in male rats receiving 240 mg/kg bw/day.

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 240 mg/kg bw/day for female rats based on no effect on systemic endpoints.

- NOAEL for reproductive and fertility toxicity was 240 mg/kg bw/day based on no effect on fertility and reproductive performance.

- The NOAEL for developmental toxicity was 240 mg/kg bw/day based on no effect on mortality, clinical signs and postnatal growth.


Justification for selection of Effect on fertility via oral route:
There is an oral study with the test substance N-butylaminoethanol available.

Justification for selection of Effect on fertility via inhalation route:

There is no inhalation study with the test substance available. However, inhalation is the relevant route of exposure for this substance. There is an oral study with the test substance N-butylaminoethanol available.


Justification for selection of Effect on fertility via dermal route:
The substance is irritating to skin therefore no study via this route is available.

Effects on developmental toxicity

Description of key information

- oral, developmental toxicity study (OECD414), rat: NOAEC(reproductive and developmental parameters; males and females) = 280 mg/kg bw/day, NOAEL (maternal toxicity) = 140 mg/kg bw/day (highest dose tested). LOAEL (maternal toxicity) = 280 mg/kg/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-05-06 - 2020-03-20 (experimental phase: 2019-06-20 - 2019-10-24)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Remarks:
Dose-range finding study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on June 25, 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan: WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 12 to 13 weeks (initiation of the acclimatisation)
- Weight at study initiation: mean: 223.16 ± 12.19 (SD), body weight range 194.6 – 256.7g (prior to acclimatisation)
- Fasting period before study: No
- Housing: Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male plus one female). Mated female rats were housed individually. During the study, rats were housed in solid floor polypropylene rat cages (size: 41cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless-steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5/6 tier racks. The bottom of the cages was layered with clean sterilised rice husk as the bedding. Cages with bedding material and water bottles were changed twice a week.Chemical and microbial contaminant analysis of samples of the bedding and enrichment materials are being checked at every six months intervals.
- Diet (e.g. ad libitum): The rats were fed ad libitum with standard rodent pelleted diet (Certified Teklad Global 16 % Protein Rodent Diet, Batch No 2016SC-010419MA, procured from Envigo, Inc., USA). Received Food: with certificate of analysis of nutrient content from the food supplier. Quality of food was monitored incompliance with JRF/TOX/SOP-2077. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Unlimited supply of drinking water in polypropylene bottles (capacity 250 mL). Drinking water was filtered through Reverse Osmosis water filtration system. Quality of water was monitored in compliance with JRF/TOX/SOP-2077. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: A total of 100 male and 120 female rats were obtained to permit the selection and/or replacement of individuals before the start of the cohabitation and acclimated in the experimental room for a period of 6 days prior to the cohabitation. During the acclimatisation period, rats were checked once daily for clinical signs of toxicity that would preclude use on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 66-68
- Air changes (per hr): 17 (June) or 16 (July)
- Photoperiod (hrs dark / hrs light): 12 hours of lighting and 12 hours of darkness
- Mean light intensity (in LUX): 207 (June), 241 (July)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantity of the test item for each dose group was received from the Test Item Control Office (TICO). Test item was formulated in the vehicle. The dose formulations were prepared every day prior to the dosing and thoroughly mixed using magnetic stirrer in order to make a homogeneous preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Reverse osmosis (RO) water was selected as a vehicle based on a solubility check performed at the JRF (JRF Study N° 410-1-02-21393) and after consultation with the Sponsor.
- Concentration in vehicle: 0, 70, 140, 280 mg/kg bw/day
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Remarks:
Concentration and homogeneity was analysed before treatment and once during treatment
Details on analytical verification of doses or concentrations:
The stability of the test item in the vehicle was established in JRF Study number 228-2-13-21392. Based on the results, it was observed that the test item in the vehicle was stable up to 8 days. Active ingredient (a.i.) concentration and homogeneity of dose formulations were analysed by the Department of Chemistry, using a validated analytical method (JRF Study number 228-2-13- 21392).
Two sets of samples (10 samples per set) were collected by sampling three aliquots (top, middle, and bottom layers) from each concentration except the vehicle control (only one aliquot from middle layer). Sampling was done before the start of treatment (July 01, 2019) and once during the treatment period (July 08, 2019) for dose formulation analyses. One set of samples was used for analyses and another set of samples was refrigerated. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis are ± 10 % from nominal value and %CV < 10. The dose formulation analyses results were included in the final report as an appendix.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until the requisite numbers of mated females were obtained
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Two weeks, from the 5th day of gestation (GD) to GD 19
Frequency of treatment:
once daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Remarks:
low dose
Dose / conc.:
140 mg/kg bw/day (actual dose received)
Remarks:
mid dose
Dose / conc.:
280 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
25 females / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the result of a prenatal developmental oral toxicity study of N-butylaminoethanol in Wistar rats (Dose Range Finding) (JRF study number 461-1-04-22168) and in consultation with the study sponsor. In dose range finding study, two mortalities, adverse clinical signs, and decrease in mean male foetal weight were observed at high dose (320 mg/kg bw/day). No effects were observed at a low dose (60 mg/kg bw/day), a mid dose - I (120 mg/kg bw/day), or a mid dose - II (240 mg/kg bw/day). Hence, following dose levels: low dose – 70, mid dose – 140, and high dose – 280 mg/kg bw/day were selected for the present study.
- Rationale for animal assignment (if not random):
Mated females were assigned to dose groups by stratified randomisation based on GD 0 body weights. Body weights of GD 0 were arranged in descending order, from the heaviest to the lightest on the first GD 0 date and assigned in the same order on the subsequent days. Beginning with the heaviest weight, one rat was randomly assigned to each group. In the event that the total number of rats mated on a given day was not an even multiple of the number of treatment groups, the mated females were assigned to complete the last incomplete stratification group and then assigned to the next stratification group until all GD 0 females for that day were assigned.

Other:
- Justification of Test System: The albino rat was selected as a test system because it has been historically shown to be a suitable model for the prenatal developmental toxicity studies. This species is also recommended by the OECD and other regulatory authorities. This species and strain of animal are recognised as appropriate for developmental toxicity studies. JRF has historical control data on the background incidence of foetal malformations and variations in the Wistar rats.
- Justification of route of administration: The route of vehicle and test item administration was gavage and was selected as per Sponsor’s recommendation.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Rats were observed daily, twice, for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the study period, visible clinical signs, such as changes in skin, fur, eye, mucous membranes, as well as the behavioural pattern of the pregnant female rats were recorded twice daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights for the mated female rats were recorded individually on days 0, 3, 5, 8, 11, 14, 17, and 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- A fixed amount of pelleted rat food was presented to each mated female rat and the food remaining at the end of the specified interval was recorded. The food consumption was determined for the following intervals: Days 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, 17-20, and 5-20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Non-gravid uteri (by staining with 5 % ammonium sulphide to confirm non-pregnant status), ovaries and gravid uteri including the cervix were removed and examined immediately. Ovaries, foetuses, and non-gravid uteri were preserved and identified. Weight of the thyroid gland was recorded from all female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in high and control dose group rats.

OTHER:
- Blood Collection: At the time of terminal sacrifice, blood (approximately 1 mL) was collected from all female rats under anesthesia (isoflurane) by orbital plexus puncture. Serum thyroid hormones (T4, TSH, and T3) were analysed from all female rats at the terminal sacrifice. Serum thyroid hormones T3 and T4 were analysed at JRF using validated bioanalytical method whereas TSH was analysed at JRF by an ELISA method.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including the cervix)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live foetuses, number of dead foetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes

The foetuses were examined for gross external anomalies and findings were recorded.
Approximately 50 % of the foetuses from each litter were subjected to the detailed visceral evaluation by microdissection. Cephalic evaluation was done by head razor sectioning after fixation in acetoalcohol formalin solution (i.e., Davidson’s fixative).
The procedure for evaluation of soft tissue was as per the JRF/TOX/SOP-279, according to Staple’s technique.

The remaining 50 % of the foetuses from each litter were subjected to the detailed skeletal evaluation after processing and staining with alizarin red. The skeletal evaluation was performed, as per JRF/TOX/SOP-232, according to Staple’s and Schnell method. The foetuses were preserved in isopropyl alcohol and glycerin solution.
Statistics:
- Data pertaining to individual rats were provided.
- All raw data were processed to determine group means and standard deviations with statistical significance between the control and treatment groups using validated statistical software.
- The parametric data (body weight, body weight change, food consumption, hormones (T3, T4, and TSH) results, and organ weight) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data do not meet the homogeneity of variance, statistical analysis was extended following a decision tree.
- The non-parametric data (mortality rate, pregnancy rate, foetal observations etc.) were analysed using Chi-square test.
- Count data (viz., foetal count, number of corpora lutea, number of implants, number of live foetuses, number of dead foetuses, number of early and late resorption) were subjected to non-parametric Kruskal-Wallis test.
- AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
- Non-pregnant rats were not subjected to statistical analysis.
- Flags for significant difference between control and treated groups (single arrow for p ≤ 0.05 and double arrows for p ≤ 0.01) were given in the table along with the footnote.
Historical control data:
JRF has historical control data on the background incidence of foetal malformations and variations in the Wistar rats.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical sign of toxicity was observed up to a dose level of 140 mg/kg bw/day.
In 280 mg/kg bw/day dose group, salivation was observed approximately 10 to 15 minutes after dosing on a few days in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding was considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and body weight changes of the pregnant female rats were comparable between the control and the test item treated groups up to a dose level of 140 mg/kg bw/day.

For the 280 mg/kg bw/day dose group, a statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. A statistically significant decrease in day 20 corrected body weight was also noted for this group. In addition, statistically significant decreases in mean body weight change were observed during the gestation days 5-8, 8-11, 17-20, and 5-20. Decreased mean body weight change for this group were also observed during the gestation period days 11-14 and 14-17 without statistical significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the pregnant female rats was comparable between the control and the test item treated groups up to a dose level of 140 mg/kg bw/day.
Statistically significant decreased mean food consumption was seen for 280 mg/kg bw/day dose group for gestation days 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.
Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test item treatment did not lead to any alteration in the absolute and relative organ weight of the thyroid gland.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Internal (gross) examination of the terminally sacrificed control and test item treated female rats did not reveal any lesion of pathological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination of the thyroid gland revealed ultimobranchial cyst/s in 2 and 1 female rats of the control and 280 mg/kg bw/day dose group, respectively, and considered as spontaneous or incidental and therefore non-adverse in nature.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Thyroid Hormone Analysis: Serum level of T3, T4, and TSH in female rats belonging to test item groups was comparable to the control group.
- External a examination of the terminally sacrificed control and test item treated female rats did not reveal any lesion of pathological significance.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups.
Dose descriptor:
LOAEL
Effect level:
280 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (actual dose received)
Basis for effect level:
other: All examined parameters
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean weight of male, female and total foetuses (male + female) was comparable between the control and the test item treated groups up to the dose level of 140 mg/kg bw/day.
In the 280 mg/kg bw/day dose group, male foetus body weight was statistically significantly decreased (- 6.57 % than control group) when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetal body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male sex ratio were comparable between the control and the test item treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean count of male, female and total foetuses (male + female) was comparable between the control and the test item treated groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
A total of 278, 264, 272, and 252 foetuses were examined in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
No external anomalies were observed in foetuses of the control and treatment groups up to the dose level of 280 mg/kg bw/day except one runt foetus (1/14) in the 280 mg/kg bwt/day dose group. This finding was comparable with that of the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 143, 137, 143, and 131 foetuses were observed for the skeletal evaluation in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
There were several incidences of the statistically significant differences in the skeletal variations in all test item treated groups (see Table 1, "Any other information on results"). These decreased skeletal variations were only because of higher number of incidences in control group compared to treatment and have no toxicological relevance. Due to the lack of dose dependency, the increase in number of foetuses and litters with Rib: Full supernumerary was considered as incidental and non-adverse (see Table 2, "Any other information on results"). In case of xiphisternum, the data indicate a delayed ossification associated with reduced foetal weights i.e., a delay in foetal development rather than a direct effect on bone tissue. In addition, this increase in the number of foetuses with Xiphisternum: Unossified was within the range of historical control data therefore considered as non-adverse in nature.
Visceral malformations:
no effects observed
Description (incidence and severity):
A total of 135, 127, 129, and 121 foetuses were examined in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
No gross treatment related visceral findings were observed in foetuses of the control and the treatment groups up to the dose level of 280 mg/kg bw/day with the exception of dilated ureter seen in one foetus from the control group. There were no any other visceral findings in any dose groups.
Other effects:
no effects observed
Description (incidence and severity):
The mean anogenital distance of male and female foetuses were comparable between the control and the test item treated groups
Dose descriptor:
NOAEL
Effect level:
280 mg/kg bw/day (actual dose received)
Basis for effect level:
other: all examined parameters
Abnormalities:
no effects observed
Developmental effects observed:
no
Lowest effective dose / conc.:
280 mg/kg bw/day (actual dose received)
Treatment related:
no

Table 1: Statistically significant decrease in skeletal variations

 Group  G1  G2  G3  G4
 Dose (mg/kg b. bw/day)  0 70   140  280
 Total N° of Foetuses Examined  143  137  143 131 
 14th Rib: Extra ossification centre  63  35 (decrease)   38 (decrease)   34 (decrease)
 Rib: Short supernumerary  33   17 (decrease)   19 (decrease)   18 (decrease)
 Squamosal: Incomplete ossification  7   1 (decrease)   1 (decrease)   0 (decrease)
 Total N° of Litters Examined  23  23  22  22
 Interparietal: Incomplete ossification  6  2   1 (decrease)  0 (decrease)
 Squamosal: Incomplete ossification  6   1 (decrease)   1 (decrease)   0 (decrease)

Table 2: Statistically significant increase in skeletal variations

 Group  G1  G2  G3  G4
 Dose (mg/kg bw/day)  0 70   140  280
 Total N° of Foetuses Examined  143  137  143 131 
Rib: Full supernumerary 1 3 10 (increase) 0
Xiphisternum: Unossified 0 5 (increase)  8 (increase)  13 (increase)
Total N° of Litters Examined  23  23  22  22
 Rib: Full supernumerary  1  3   6 (increase)  0
Conclusions:
Based on the result of the present study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of N-butylaminoethanol for maternal toxicity is 140 mg/kg bw/day and 280 for developmental toxicity under the conditions and procedures followed in the study. The LOAEL for maternal toxicity was 280 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.
Executive summary:

Summary: This study was performed to evaluate the prenatal developmental and maternal toxicity potential of N-butylaminoethanol, when administered orally through gavage to mated female rats.

Method: N-butylaminoethanol was administered orally from gestation days (GD) 5 to 19, through gavage, to 25 mated female rats per group at dose levels of 70, 140, and 280 mg/kg bw/day. The control group received the vehicle, reverse osmosis water, only. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period. All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised and the number of corpora lutea counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid stimulating hormone) were analysed from all female rats during the terminal sacrifice. At the time of terminal sacrifice, the weight of the thyroid gland was recorded from all female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in control and high dose group female rats. Following appropriated fixation, foetuses were examined for visceral abnormalities including razor sectioning of the head, and for skeletal abnormalities.

Dose formulations were analysed for the test item concentration and homogeneity, prior to the initiation of the treatment and once during the treatment period.

Results: The dose formulation analysis results revealed that the mean recovery was within the acceptance level of ± 10 % of nominal value and % CV was < 10. The dose formulation analysis demonstrated that the dose formulations were homogeneous.

No treatment related effects were seen in the 70 and 140 mg/kg bw/day groups for any evaluated parameter which include: Mortality and clinical signs of toxicity, mean body weight, body weight change, food consumption, and 20th day corrected body weight, Mean absolute and relative uterine weights, serum level of T3 (Liothyronine), T4 (Levothyroxine), and TSH (Thyroid Stimulating Hormone), external and internal (gross) examination of the terminally sacrificed female rats, absolute and relative weights of the thyroid gland, Histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count and foetal body weight of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, male sex ratio, Incidences of malformation/variation for external, visceral, head razor and skeletal examination.

No treatment related adverse effects were seen in the 280 mg/kg b. wt./day group for following parameters: Mortality, salivation was observed approximately 10 to 15 minutes after dosing in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding is considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature, Mean absolute and relative uterine weights, Serum level of T3, T4, and TSH, External and internal (gross) examination of the terminally sacrificed female rats, Absolute and relative weight of thyroid gland, histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, statistically significant decrease in mean body weight of male foetuses (- 6.57 % than control group) was observed when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetus body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature, Male sex ratio, Incidence of malformation/variation for external, visceral, head razor, and skeletal examination of foetuses.

The following treatment related adverse effects were seen in the 280 mg/kg bw/day group:

- Statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. Statistically significant decrease in 20th day corrected body weight of the pregnant female rats was observed. Statistically significant decrease in the mean body weight change of the pregnant female rats was observed during the gestation period 5-8, 8-11, 17-20, and 5-20. Decrease in the mean body weight change of the pregnant female rats was also observed during gestation period 11-14 and 14-17 without statistical significance.

- Statistically significant decrease in mean food consumption of the pregnant female rats was observed during the gestation period 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.

- Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.

Conclusion: Based on the result of the present study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of N-butylaminoethanol for maternal toxicity is 140 mg/kg bw/day and 280 for developmental toxicity under the conditions and procedures followed in the study. The LOAEL for maternal toxicity was 280 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
280 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study (Klimish 1).
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

There is an oral developmental dose toxicity study available for N-butylethanolamine. This study was performed to evaluate the prenatal developmental and maternal toxicity potential of N-butylaminoethanol, when administered orally through gavage to mated female rats. N-butylaminoethanol was administered orally from gestation days (GD) 5 to 19, through gavage, to 25 mated female rats per group at dose levels of 70, 140, and 280 mg/kg bw/day. The control group received the vehicle, reverse osmosis water, only. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period. All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised and the number of corpora lutea counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid stimulating hormone) were analysed from all female rats during the terminal sacrifice. At the time of terminal sacrifice, the weight of the thyroid gland was recorded from all female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in control and high dose group female rats. Following appropriated fixation, foetuses were examined for visceral abnormalities including razor sectioning of the head, and for skeletal abnormalities.

Dose formulations were analysed for the test item concentration and homogeneity, prior to the initiation of the treatment and once during the treatment period.

As results the following was reported: the dose formulation analysis results revealed that the mean recovery was within the acceptance level of ± 10 % of nominal value and % CV was < 10. The dose formulation analysis demonstrated that the dose formulations were homogeneous.

No treatment related effects were seen in the 70 and 140 mg/kg bw/day groups for any evaluated parameter which include: Mortality and clinical signs of toxicity, mean body weight, body weight change, food consumption, and 20th day corrected body weight, Mean absolute and relative uterine weights, serum level of T3 (Liothyronine), T4 (Levothyroxine), and TSH (Thyroid Stimulating Hormone), external and internal (gross) examination of the terminally sacrificed female rats, absolute and relative weights of the thyroid gland, Histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count and foetal body weight of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, male sex ratio, Incidences of malformation/variation for external, visceral, head razor and skeletal examination.

No treatment related adverse effects were seen in the 280 mg/kg b. wt./day group for following parameters: Mortality, salivation was observed approximately 10 to 15 minutes after dosing in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding is considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature, Mean absolute and relative uterine weights, Serum level of T3, T4, and TSH, External and internal (gross) examination of the terminally sacrificed female rats, Absolute and relative weight of thyroid gland, histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, statistically significant decrease in mean body weight of male foetuses (- 6.57 % than control group) was observed when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetus body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature, Male sex ratio, Incidence of malformation/variation for external, visceral, head razor, and skeletal examination of foetuses.

The following treatment related adverse effects were seen in the 280 mg/kg bw/day group:

- Statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. Statistically significant decrease in 20th day corrected body weight of the pregnant female rats was observed. Statistically significant decrease in the mean body weight change of the pregnant female rats was observed during the gestation period 5-8, 8-11, 17-20, and 5-20. Decrease in the mean body weight change of the pregnant female rats was also observed during gestation period 11-14 and 14-17 without statistical significance.

- Statistically significant decrease in mean food consumption of the pregnant female rats was observed during the gestation period 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.

- Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.

Conclusion: Based on the result of the present study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of N-butylaminoethanol for maternal toxicity is 140 mg/kg bw/day and 280 for developmental toxicity under the conditions and procedures followed in the study. The LOAEL for maternal toxicity was 280 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.

Justification for selection of Effect on developmental toxicity: via oral route:

There is an oral study with the test substance N-butylaminoethanol available.


Justification for selection of Effect on developmental toxicity: via inhalation route:

There is no inhalation study with the test substance N-butylaminoethanol available. However, inhalation is the relevant route of exposure for this substance. There is only an oral study with the test substance N-butylaminoethanol available.


Justification for selection of Effect on developmental toxicity: via dermal route:

There is no dermal study with the test substance N-butylaminoethanol available. Furthermore, this is not the relevant route of exposure for this substance.

Justification for classification or non-classification

The test substance N-butylamoinoethanol did not cause any adverse effect with regard to reproductive developmental parameters. Based on these data, the substance does not need to be classified and labelled for reproductive or developmental effects according to Regulation (EC) No.1272/2008.