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Repeated dose toxicity: oral

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Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-09 - 2020-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
14-day study (Dose-range finding study)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-11-06 - 2019-04-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
DRF Study in accordance with GLP. The study design was based on the study objectives and inspired from OECD Guideline 407.
Reason / purpose:
reference to other study
Remarks:
Main study
Reason / purpose:
reference to other study
Remarks:
Main study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on October 03, 2008.
Deviations:
yes
Remarks:
The study design was based on the study objectives and inspired from OECD 407
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Wistar strain was used because of the historical control data available at this JRF laboratory.
Sex:
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: 8 - 9 weeks old
- Weight at study initiation: body weight variation among rats was within ± 20 % of the mean body weight for each sex.
- Housing: Rats were housed in groups of 2 or 3 rats/cage/sex during the study period in sterile solid floor polypropylene rat cages (size: 41 × 28.2 × 18 cm). Each cage was fitted with a stainless steel (grille) top having provision for keeping rodent pellet feed and a polypropylene water bottle with a stainless steel drinking nozzle. Cages were placed in multi-tier racks (5 x 5) to ensure similar environmental conditions for all groups. The cage bottom was layered with clean sterilised rice/paddy husk as bedding material. Rats were offered environmental enrichment materials, viz. wooden chew blocks, in each cage. Cages with bedding material, enrichment material and water bottles were changed a minimum twice per week. Chemical and microbial contaminant analyses of bedding material samples were checked at six monthly intervals
- Diet (e.g. ad libitum): Experimental rats were fed ad libitum with Teklad Certified Global 16 % protein rodent maintenance feed, Envigo USA.
- Water (e.g. ad libitum): Rats were provided Reverse Osmosis (RO) water ad libitum (RO water filtration system) in polypropylene bottles.
- Acclimation period: for a minimum period of 6 days prior to the start of dosing. During the acclimatisation period, all rats were observed once daily for clinical signs, and twice daily for morbidity and mortality.

DETAILS OF FOOD AND WATER QUALITY: Every feed consignment received is accompanied by a certificate of analysis of nutrient content from the feed supplier. The quality of feed and water was monitored.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 64-66
- Air changes (per hr): 21
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness
- mean light intensity (LUX): 221-225
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected to simulate the conditions of potential human exposure.
Vehicle:
water
Remarks:
Reverse osmosis (RO) water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the vehicle to make the dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing. The dose formulations were prepared each day of dosing and were administered to rats immediately after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Reverse osmosis (RO) water was selected as vehicle based on solubility check
- Concentration in vehicle: 0, 40, 125, 400 mg/kg bw/day
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of test item in vehicle, concentration and homogeneity of dose formulations were analysed.
Two set of samples (10 samples per set) were collected by sampling three aliquots (top, middle and bottom layers) from each concentration and one aliquot (from the middle layer) for the vehicle control. Sampling was performed before the start of treatment and once during the treatment period for dose formulation analyses. The mean concentration was determined and compared to the nominal value and the coefficient of variation was calculated. The acceptance criteria used for analysis were ± 10 % from nominal value and % CV <10.
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on available literature where LD50 of test item was 890 mg/kg bw, high dose was selected with aim that it should produce adverse effect whereas mid and low dose levels should not produce any adverse effect. Hence, three dose levels; low 40, mid 125 and high dose 400 mg/kg bw/day were selected.
- Rationale for animal assignment (if not random): random
- Fasting period before: Rats were fasted overnight (with ad libitum supply of drinking water) prior to sacrifice.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice a day for mortality and signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During acclimatisation, all rats were observed once daily for clinical signs. All rats were observed twice a day for any visible clinical signs during treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded for individual rats on day 1 (before commencement of treatment), and on days 4, 8, 11 and 14. Rats were also weighed at death and on the day of necropsy (fasted body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- A weighed amount of feed was offered to rats in each cage on days 1, 4, 8 and 11 and the leftover feed was measured on days 4, 8, 11 and 14.

OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Rats were examined carefully for external abnormalities. The thoracic and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect any abnormalities.

HISTOPATHOLOGY: Yes
- wet weight of organs recorded for the liver, kidneys, adrenals, testes, epididymis, prostate, cowper's gland, levator ani plus, bulbocavernosus muscles (LABC), seminal vesicles with coagulating glands, thyroid, thymus, heart, brain, spleen, uterus with cervix, and ovaries.
Statistics:
- All the parameters characterized by continuous data were subject to Shapiro-Wilk's test for normality checking, followed by Barlett's test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett's t-test.
- When the data did not meet the normality, data was transferred to log form and square-root to check the normality again, then the Kruskal-wallis test was performed to calculate significance.
- When the data did not meet the homogeneity of variance, Student s t-test was performed to calculate significance.
All analyses and comparisons were evaluated at the 5% (P 0.05) and 1% (P 0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
In vehicle control, low and mid dose groups, all male and female rats were normal throughout the study period.
Description (incidence):
- No morbidity and mortality was observed in rats from vehicle control, low and mid dose groups.
- One mortality (Rat N° 32 on day 7) was observed in male rat belonging to high dose group. Two rats (one male and one female rat) were sacrificed under moribund condition (one on day 13 Rat N° 31; another on day 8 Rat N° 40) in high dose group. The rats sacrificed under moribund condition and mortality observed in high dose group could be considered as treatment related adverse effect as it was observed after severe clinical signs like dyspnoea, gasping and rales.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- In male rats belonging to high dose group, statistically significant decrease in body weight was observed on treatment day 4, 8, 11 and 14 as compared with that of the vehicle control group.
- Despite of statistical insignificance, decrease in body weight gain throughout treatment period was observed in male rats belonging to high dose group as compared with that of the vehicle control group.
- The body weight of all female rats treated with test item were comparable with that of the vehicle control group.
- In continuation, despite of statistical insignificance, decrease in body weight gain during treatment days 1-4 and 4-8 was observed in female rats belonging to high dose group as compared with that of the vehicle control group. Statistically significant decrease in body weight gain was observed during treatment days 11-14 in female rats belonging to high dose group as compared with that of the vehicle control group. The changes observed in high dose male and female rats could be considered as treatment related adverse in nature based on observed mortality, toxic clinical signs and concurrent decrease in food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in food consumption during treatment days 1-4 in female rats belonging to high dose group was observed as compared with that of the vehicle control group. The changes observed in high dose group male and female rats could be considered as treatment related adverse in nature based on the concurrent decrease in body weight.
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
Description (incidence and severity):
- Terminal body weight, absolute and relative organ weight of low and mid dose groups in male rats was comparable, except statistically significant decrease in seminal vesicle with coagulating gland (SV with CG) was observed in mid dose group as that of the vehicle control group.
- Statistically significant decrease in terminal body weight, absolute weight of heart in male rats belonging to high dose group was observed as compared with that of the vehicle control group. Statistically significant increase in relative weight of kidney in male rats belonging to high dose group was observed as compared with that of the vehicle control group.
- Terminal body weight, absolute and relative organ weights of female rats treated with the test item were comparable to that of the vehicle control group. The decrease in SV with CG weight in mid dose groups could be considered as incidental in nature due to lack of dose dependent effect. Decrease in terminal body weight could be considered as treatment related as same changes were seen in body weight and body weight gain. Decrease in absolute weight of heart in high dose group might be treatment related, but the toxicological significance was not concluded under this study with limited information.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- External examination of terminally sacrificed rats belonging to all groups (G1, G2, G3, and G4) did not reveal any abnormality. Rats sacrificed under moribund condition of either sex and found dead male rat did not reveal any abnormality.
- Internal examination of terminally sacrificed rats belonging to all groups (G1, G2, G3, and G4) did not reveal any abnormality. Rats sacrificed under moribund condition and found dead male rat did not reveal any abnormality. Female rat which was sacrificed under moribund condition had stomach & intestine gaseous distension.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Conclusions:
Based on the results, the following dose levels are suggested for the further long-term toxicity study (in mg/kg bw/day):
Option Low Dose Mid Dose High Dose
1 70 140 280
2 75 150 300
3 62.5 125 250
4 60 120 240

Option 4 was selected for further repeated dose study.
Executive summary:

Study objective: The objective of this study was to evaluate the potential toxicity of n-butylaminoethanol in Wistar rats following daily oral (gavage) administration for at least 14 consecutive days. The results will be used for the safety assessment of the test item and will be used to select dose levels for possible further long-term toxicity studies. This study was conducted in compliance with OECD Principles of GLP (1998).

Method: Prepared dose formulations of the test item in the vehicle (reverse osmosis water) were administered by gavage for a period of 14 consecutive days at three graduated dose levels to male and female rats. A concurrent vehicle control group of male and female rats received the vehicle only. During the experimental period, all rats were observed for clinical signs, morbidity and mortality. Body weight was recorded for each rat and food consumption was calculated for each cage twice a week. At the end of the treatment period, all surviving rats were sacrificed and subject to gross pathological examination. Organ weights were recorded for each terminally sacrificed rat.

Results: All doses were within the acceptable range of ± 10 % of nominal concentration of the %CV was less than 20 which suggests that the prepared dose formulation had acceptable a.i. concentration and were homogenously prepared.

At 40 and 125 mg/kg bw/day: no mortality or clinical signs, no effects on body weight or body weight gain, food consumption and organ weight (absolute and relative), no abnormalities (external or internal).

At 400 mg/kg/bw/day: one mortality (male) on day 7 and two (male on day 13 and female on day 8), clinical signs like lethargy dyspnoea, abdominal breathing, gasping, rales piloerection, weakness, vocalisation and writhing in males (2 of 5) and females (3 of 5). Treatment related decrease in body weight, body weight gain, food consumption, terminal body weight and organ weight (absolute heart) were observed in male rats. No effects on body weight, terminal body weight and organ weight (absolute and relative) on female rats. No abnormalities (external or internal).

Conclusion: Based in results, 60, 120, 240 mg/kg bw/day were selected for further repeated dose study.

Reason / purpose:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
exposure period >= 50 days
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-01-12 - 2020-01-28
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: 11-13 weeks
- 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.
- Diet (e.g. ad libitum): Rats were fed ad libitum with standard rodent diet.
- Water (e.g. ad libitum): Rats were provided reverse osmosis (RO) water ad libitum (from a RO water filtration system) in polypropylene bottles.
- Acclimation period: 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.

DETAILS OF FOOD AND WATER QUALITY: The quality of food and water was monitored.

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 oral 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
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). Total number of treatment days was 50
- Female rats were dosed during pregnancy and up to post-partum day 14.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
240 mg/kg bw/day (actual dose received)
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.
Observations and examinations performed and frequency:
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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the time of terminal sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes (Rats were deprived of food overnight (but allowed water ad libitum) prior to blood collection)
- How many animals: from all rats (except female sacrificed on GD 25)
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the time of terminal sacrifice
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: NBO (Home Cage Observation, Handling Observation, Open Field Observation) were evaluated prior to initiation of treatment and at weekly intervals thereafter
- Dose groups that were examined: FOB and NBO were performed in last week of sacrifice using five randomly selected rats/sex/group towards the end of the study
- Battery of functions tested: FBO: sensory activity / grip strength / motor activity / other: Hind Limb Foot Splay

OTHER: THYROID HORMONE ANALYSIS
Serum thyroid hormones (T4, TSH, and T3) levels were analysed from parental male rats, parental female rats and terminally sacrificed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY: Yes
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).
Statistics:
Data 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.
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.
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.
- 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.
- 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 comparable.
- Handling Observations: NBO performed during handling of rats did not reveal any abnormality related to treatment.
- Open Field Observations: 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 sign 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, sensory reactivity parameters, the hindlimb and forelimb grip strength values, 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
Description (incidence and severity):
- Statististically significant decreases were noted in absolute weights of heart, adrenals and prostate in high dose male rats. These findings were considered related to the lower body weights for the high dose males as relative weights were consistent with control. Additionally, no histomorphologic changes were seen in these organs.
- The statistically significant increase in relative weight of testes in high dose male, was related to the lower terminal body weight.
- A statistically significant increase was noted in weight of thymus in low dose male (absolute and relative), mid and high dose male (relative). Effects were not considered as test item related in absence of dose dependency (more severity in low dose than the high dose despite a 4-fold difference in dose levels).
- A statistically significant decrease was noted in weights of seminal vesicles with cogulating gland in low dose and mid dose (absolute and relative), and high dose (absolute). Effect was not related to test item treatment due to absence of dose dependency (more severity in low and mid dose than the high dose).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Internal examination of rats of either sex in groups (G1 to G4) did not reveal any abnormality except for; enlarged kidneys in rat N° 41 (G2 male) and bilateral enlarged epididymides in rat N° 94 (G4 male). The lesions were considered as incidental/spontaneous.
Neuropathological findings:
not examined
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.
- 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.
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: systemic endpoints
Conclusions:
Based on results of this study, it was concluded that: the 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. The 240 mg/kg bw/day was the NOAEL for female rats based on no effect on systemic endpoints.
Executive summary:

The study was conducted to evaluate the toxic characteristics, systemic toxicity and neurotoxicity of N-Butylaminoethanol when administered by gavage to male and female Wistar rats over 28 days and beyond (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, neurobehavioural observation (NBO), functional observational battery (FBO), 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 sign, mortality, body weight, body weight gain, feed consumption, NBO, FOB, clinical pathology, and organ weight (absolute and relative) were observed. No treatment related macroscopic and microscopic lesions were observed.

High dose:

- No sign of toxicity on mortality, NBO, FOB, organ weight, clinical pathology (absolute and relative) was observed. No treatment related macroscopic and microscopic lesions were observed.

- Salivation was 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 effect on endocrine sensitive endpoints

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. The 240 mg/kg bw/day was the NOAEL for female rats based on no effect on systemic endpoints.

Reason / purpose:
reference to other study
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on June 25, 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Butylaminoethanol
- Appearance: Colorless liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan: WIST
Details on species / strain selection:
The albino rat was selected as a test system because it has been historically shown to be a suitable model for repeated dose toxicity studies and is recommended by the OECD and other regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundataion, Animal Breeding Facility (ABF)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks old
- Weight at study initiation: body weight variation among the rats were within ± 20 % of the mean body weight for each sex
- Fasting period before study:
- Housing: Rats were housed in groups of 2 rats/sex/cage during the study period in sterilised solid floor polypropylene rat cages while rats having more than 500 g body weight were housed individually in separate cage after completion of 8 week of treatment. Each cage was fitted with a stainless-steel top grille with provision for rodent pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Rats were provided with wooden chew blocks as an environmental enrichment material in each cage. Water bottles were changed daily. Cages, bedding and enrichment materials were changed on alternative days thereafter.
- Diet (e.g. ad libitum): standard rodent pellet food ad libitum (Teklad Certified Global 16% Protein Rodent Diet, Envigo, U.S.A.)
- Water (e.g. ad libitum): unlimited supply of clean and filtered drinking water (RO water filtration system) in autoclaved polypropylene bottles
- Acclimation period: 5 days prior to randomisation

DETAILS OF FOOD AND WATER QUALITY: Every food consignment received at JRF is accompanied by a certificate of analysis of nutrient content from the supplier. Quality of food (microbial contaminant) and water (microbial and chemical contaminant) is routinely checked.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 65 - 67
- Air changes (per hr): 20 - 21
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The route of vehicle and test item administration was oral through gavage and was selected as per ECHA test order.
Vehicle:
water
Remarks:
Reverse osmosis (RO)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared daily and thoroughly mixed using a magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulation was given to rats immediately after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on solubility test
- Concentration in vehicle: 0, 60, 120, 240 mg/kg bw/day
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability, active ingredient concentration, and homogeneity of test item in dose formulation were analysed.
On the basis of stability results, the test item was found stable up to 7 days in dose formulation.

Two set of samples (total 20 samples) were collected by sampling three aliquots from each concentration (upper, middle and lower layers) and one sample (from middle layer) from vehicle control on days 1, 29, 57 and 85. One set of samples (total 10 samples) were used for analyses.
The mean concentrations were determined and compared to the nominal value and the coefficient of variation was calculated. The acceptance criteria used for analysis was:
- Dose formulation concentration: ± 10 % from nominal value
- Homogeneity of mixing: %CV< 10
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats / sex / group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Three dose levels (low dose - 60, mid dose - 120 and high dose - 240 mg/kg b. wt./day) were selected based on results of 14 day dose range finding study and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study. In 14-day DRF study, three dose levels (40, 120, and 400 mg/kg bw/day) were evauated. In this study, 3 mortalities were observed with toxic clinical sign at 400 mg/kg bw/day and no sign of toxicity was observed at 125 and 40 mg/kg bw/day.
In Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study, three dose levels (60, 120, and 240 mg/kg bw/day) were evaluated. At 240 mg/kg bw/day treatment related decrease in body weight, body weight gain and overall food consumption was observed in male rats. No treatment related changes were observed at dose level 60 and 120 mg/kg bw/day. Considering the effects at 240 mg/kg bw/day, decrease in body weight, body weight gain and overall food consumption observed in male, this dose is further selected for OECD 408 study as hightest tested dose i.e. according to the OECD 408 guideline, the highest dose level should be chosen with the aim to induce some toxicity. Hence, based on DRF and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study results, dose levels were selected.
- Rationale for animal assignment (if not random): all rats to be randomised was within the range of ± 20 % of mean body weight.
- Fasting period before blood sampling for clinical biochemistry: Rats were fasted overnight (with ad libitum supply of drinking water), prior to the blood collection (approximately 3 mL blood).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice dailyfor mortality and signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily for clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual rat body weight, of all rats, was recorded at the beginning of the treatment and weekly, thereafter. Rats from all groups were also weighed on the day of necropsy (fasted body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- A weighed amount of food was offered weekly. Food remaining was measured in each cage once weekly, during the study period.

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

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations and dose groups that were examined: Ophthalmological examination was performed on each rat with the aid of ophthalmoscope - once before commencement of the treatment and second time before the terminal sacrifice. In order to facilitate easy examination of the interior part of the eye, homatropine (2 %) eye drops were used to dilate the pupil. This mydriatic solution was instilled into the eye 20 minutes before the eye examination

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (feed was withheld but water was provided ad libitum)
- Parameters checked in table [No.3] were examined.

IMMUNOLOGY: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the initiation of treatment and weekly, thereafter
- Dose groups that were examined: each rat
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Landing Hind Limb Foot Splay

OTHER:
Neurobehavioural Observation (NBO): Home Cage Observations, Observations during Removal and Handling, Open Field Measurements
Serum thyroid hormones (T3, T4) were analysed using a validated bioanalytical method and TSH was analysed by ELISA methods.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On the day of necropsy, vaginal smear was examined to determine the stage of the oestrous cycle. All rats were examined carefully for external abnormalities. The thoracic and abdominal cavities were opened, and a thorough examination of the organs was carried out to detect abnormalities. Descriptions of all macroscopic abnormalities seen at necropsy were recorded. The following organs and tissues as applicable, from all surviving male and female rats were collected, weighed, and preserved, while from the found dead rat were collected and preserved: Adrenals, Brain, Epididymides, heart, kidneys, liver, ovaries, pituitary gland, spleen, Prostate + seminal vesicles with coagulating glands, thymus, testes, thyroid with parathyroid, uterus with cervix.

HISTOPATHOLOGY: Yes
Adrenals, Brain, Epididymides, heart, kidneys, liver, ovaries, pituitary gland, spleen, Prostate + seminal vesicles with coagulating glands, thymus, testes, thyroid with parathyroid, uterus with cervix, aorta, bone with marrow (femur with joint), oesophagus and trachea, eyes, gross lesions, lungs, lymoh nodes (prescapular, mesenteric, large intestine, mammary glands, pancreas, stomach, small intestine, skin, sciatic nerves skeletal muscle, sceletal glands, spinal cord at three levels - cervical, mid thoracic and lumbar, urinary bladder, vagina. In addition, all gross lesions were examined
Statistics:
All parameters characterised by continuous data, data were subjected to Shapiro-wilk’s test for normality (where applicable) followed by the Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s test (Gad, S.C. and Weil, C.S., 2007). When data did not meet the normality, data were transformed (log form, square root etc.) to check the normality again. When transformed data did not meet the normality, non-parametric tests were performed to calculate significance.

NBO parameters (urination, defecation, rear, and vocalisation) and FOB parameters (motor activity) data were subjected to non-parametric tests to calculate significance. When the data did not meet the homogeneity of variance, F-test was performed followed by the t-tests to calculate significance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All rats were normal, throughout the dosing period, in control, low, and mid dose groups. Mild salivation in male and female rats and gasping in male rats of high dose treated group were observed. Salivation observed in high dose group was considered a response to the dose solution and considered as non-adverse in nature. Gasping was observed only in male rats of high dose treated group. Gasping was seen in 3 out of 10 male rats in high dose group and there were no microscopic changes observed in respiratory organs. Hence, it was considered as possibly related an irritancy response to the test material and non-adverse in nature. Additionally, lethargy and weakness were observed on day 84 in the female rat N° 75 of high dose. Rat N° 75 was found dead after showing lethargy and weakness, 1 day prior.
Mortality:
no mortality observed
Description (incidence):
No mortality or morbidity were observed during the study period in the control, low, and mid dose groups. One female rat from the high dose group (rat N° 75) died on day 85 of dosing after showing clinical signs like, lethargy and weakness. However, there were no treatment related changes observed in body weight, food consumption, clinical pathology, gross and microscopic examination, in female rats of high dose group. Hence, this death was considered not to be related to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant difference in mean body weights were noted for the treated male and female groups when compared to the same sexed controls.
The mean body weight change in low dose of male rats and all treatment groups of female rats, was comparable with that of the vehicle control group. The body weight gains for mid dose male rats were statistically significantly lower than control for weeks 7 through 13. Similarly, body weight gains for the high dose male group showed statistically significantly lower body weight gains during week 2, 4, and 6 with numerically lower body weight gains at other intervals. In the absence of statistically significant changes in body weights for mid and high dose male rats and no effects on body weight or body weight gain in female rats, the lower body weight gains for the mid and high dose males are not considered an adverse effect of test item administration.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption of rats from the treatment groups were comparable with that of the vehicle control group, except statistically significant decrease in the food consumption observed during the week 3 in male rats of high dose group. This reduction in food consumption was marginal and inconsistent. Hence, it was not considered as treatment related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination did not reveal any abnormalities.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes in the haematology parameters were observed in male and female rats of the treatment groups.
- Platelet counts for high dose male and female groups were statistically significantly higher than those of the same-sexed vehicle control group. The increases were minimal and considered to be incidental in nature and non-treatment related.
- Statistically significant increases were noted in RBC and HCT in low, mid and high females. Effect lacked dose dependency and consistency between sexes and all values of all parameter so treated females were within historical range. Therefore the effect was not related to test item treatment.
- Statistically significant increase was noted in reticulocytes in high dose male, which was not related to treatment as decrease in red cell mass (RBC, haemoglobin and haematocrit) and it also lacked consistency between sexes. Moreover, values of all male rats of high dose were within historical ranges.
- Statistically significant decrease was noted in APTT in high dose male which was not considered as toxicologically significant.
- Statistically significant decrease was noted in reticulocytes in mid dose female, which was considered unrelated to test item treatment due to lack of dose dependency.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes in the clinical chemistry parameters was observed in male and female rats of the treatment group.
- Statistically significant decreases were noted in albumin in high dose male and female, and total protein in high female. The lower values were minimal in nature and, in general, were within the historical control range and no other clinical chemistry changes related to liver function were noted for this high dose group. The lower total protein and albumin values were considered incidental in nature and not treatment related.
- Statistically significant increases were noted in LDL and inorganic phosphorus in high dose males, while decrease was noted in chloride in high dose females, which were considered unrelated to test item treatment, due to lack of consistency between sexes. Moreover, though values of 3 males of high dose were above historical ranges, it was considered as associated with false elevation due to increase in platelets (Lutomski and Bower, 1994). Similarly, in high dose females only one rat had values below historical range, while historical range for LDL is not available.
- Statistically significant increases were observed in glucose, urea and BUN in mid dose males, and HDL and LDL in mid dose females, which were considered unrelated to test item treatment, due to lack of dose dependency.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urine analysis parameters did not reveal any alteration related to treatment.
- Statistically significant increase was observed in volume and pH in males of high dose animals, which was considered unrelated to test item treatment, due to lack of consistency between sexes and normal kidneys function tests (serum creatinine, urea/BUN concentration and histopathology of kidneys)
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Neurobehavioural Observations:
Home Cage Observations and Handling Observations: no abnormalities.
Open Field Observation:
- Statistically significant increase in rearing count (week 12, male rats, mid dose)
- Statistically significant increase in the urination count (week 3, male rats, low, mid, high dose)
- Statistically significant decrease in the urination count (week 2, female rats, high dose) and increase in the urination count (week 6 female rats, mid dose)
- Statistically significant increase in the defecation count (week 2, male rats, high dose)
These findings were stand-alone findings and were not supported by any other neurobehavioural parameters and motor function findings in the high dose group. Hence, the finding were not considered treatment related.

Functional Observational Battery (FOB):
- Motor Activity: The motor activity data of rats from the treatment groups were comparable with that of the control group except statistically significant decrease in the fine activity during 21-30 minute interval for high dose females. These changes were not supported with rearing count or any other neuromuscular activity parameters. Hence, this finding was considered incidental in nature.
- Sensory Reactivity Observations, Grip Strength, Hindlimb Foot Splay: Treatment groups were comparable with control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The test item did not produce any treatment related alteration in the absolute organ weights.
- Statistically significantly higher relative weights of liver for mid and high dose males, and thyroid with parathyroid for high dose males were noted. These changes were considered to be related to the lower terminal body weights noted for these groups and not a treatment effect.
- Statistically significant increase was also noted in relative weights of kidneys in mid dose male animals, which was considered unrelated to test item treatment, due to absence of dose dependency.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination (external and internal) of rats of either sex across various groups (G1 to G4) did not reveal any abnormality except for a single mid dose male (Rat No 45) which showed bilateral reduced size of testes and epididymides considered spontaneous or incidental in nature and not related to the test item treatment.
A gross pathological examination of the found dead rat No 75 revealed a generalised autolysis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The test item treatment did not lead to any alteration in the microscopic examination.
An examination of the found dead rat No 75 revealed varying degrees of autolytic changes in various organs. In the absence of any significant gross and histopathological observation, cause of death of the rat could not be defined and relation of death with the test item treatment could not be established.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Hormone Analysis: Hormone level (TSH, T3 and T4) of male and female rats of the treatment groups was comparable with that of the vehicle control group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: Hormone analysis

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
- Based on above results, it is concluded that N-Butylaminoethanol did not produce any toxicity or adverse effect up to 240 mg/kg bw/day when administered orally through gavage for 90 consecutive days in Wistar rats.
- Therefore, the No Observed Adverse Effect Level (NOAEL) of N-Butylaminoethanol is 240 mg/kg bw/day under the conditions and procedure followed in this study.
Executive summary:

This study was conducted to determine the adverse effects occurring as a result of the daily repeated oral (gavage) administration of N-Butylaminoethanol for a period of 90 consecutive days in Wistar rats.

Method:

 Dose levels

G1 (vehicle 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

Number of rats

 10 rats/sex/group

 Vehicle  RO Water

Based on the results from a stability study, the test item was found to be stable for 7 days in dose formulation, using RO water as the vehicle. Three samples of dose formulation (upper, middle, and lower layers) from each concentration and one sample from the vehicle control (middle layer) were collected on days 1, 29, 57, and 85 of dosing, for homogeneity and active ingredient (a.i.) concentration analysis.

Each rat was observed twice daily for clinical signs, morbidity, and mortality, during the treatment period. Body weights were recorded weekly for all rats. Body weight change and food consumption were calculated weekly. Neurobehavioural observation (NBO) was performed on all rats, prior to the initiation of the treatment and weekly, thereafter. The ophthalmological examination was performed on all rats from each group, prior to the initiation of the treatment and on surviving rats, prior to the terminal sacrifice. A Functional Observational Battery (FOB) was performed on all rats, during the 12th week of treatment. Haematological and clinical chemistry analyses were performed at the end of the treatment. Urine was collected from all surviving rats, at the end of the treatment for urine analysis.

All surviving rats were sacrificed by carbon dioxide asphyxiation at end of the treatment period. All surviving and found dead rats were subjected to a full gross necropsy. On the day of necropsy, vaginal smears were examined in all surviving female rats to determine the stage of estrous cycle. Absolute organ weights were recorded, and relative organ weights were calculated for the adrenals, brain, epididymides, heart, kidneys, liver, pituitary gland, spleen, seminal vesicles with coagulating glands and prostate, thymus, testes, thyroid with parathyroid and uterus with cervix. Detailed histopathological examination was carried out in all rats from the vehicle control and high dose groups.

Results:

Results of the active ingredient concentration and homogeneity analysis of the dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable ranges of ± 10 % of the nominal concentration.

Mortality was observed in one high dose female rat on day 85. However, there were no treatment related changes observed in body weight, food consumption, clinical pathology, gross and microscopic examination, in female rats of high dose group. Hence, this death was considered not to be related to treatment.

Mild salivation in male and female rats and gasping in male rats of high dose treated group were observed on multiple days of study. Salivation observed in high dose group was considered as a response to the dose solution and considered as non-adverse in nature. Gasping was not supported with any microscopic change observed in respiratory organs. Hence, it was considered as possibly related an irritancy response to the test material and non-adverse in nature.

No treatment related changes were observed of the treatment groups as compared to control during the ophthalmological examination, in the neurobehavioural observation and functional observational battery, in the mean body weight and food consumption and in the heamatology, clinical chemistry and urinalysis parameters. The test item did not produce any treatment related alteration in the terminal body weight and absolute organ weight. A gross examination (external and internal) of rats of either sex across various groups (G1 to G4) did not reveal any abnormality with the exception of a male rat from the mid dose group (Rat No 45) showed bilateral reduced size of testes and epididymides, which was spontaneous or incidental in nature and not related to the test item treatment. A gross pathological examination of the found dead rat revealed a generalised autolysis. The test item treatment did not lead to any alteration in the microscopic examination. An examination of the found dead rat No 75 revealed varying degree of autolytic change in various organs. In the absence of any significant gross and histopathological observation, cause of death of the rat could not be defined and relation of death with the test item treatment could not be established.

Conclusion:

During this study, there was no treatment related adverse change observed in the body weight, food consumption, clinical pathology, and histopathlogy, up to dose level 240 mg/kg bw/day.

Based on above results, it is concluded that N-Butylaminoethanol did not produce any toxicity or adverse effect up to 240 mg/kg bw/day when administered orally through gavage for 90 consecutive days in Wistar rats. Therefore, the No Observed Adverse Effect Level (NOAEL) of N-Butylaminoethanol is 240 mg/kg bw/day under the conditions and procedure followed in this study.