2-butylaminoethanol

Endpoint summary

Description of key information

The substance N-butylethanolamine (BEA) did not induce mortalities and clinical signs in treated animals in a 90 day oral repeated dose toxicity study. Furthermore, BEA was shown not to induce reprotoxic or developmental toxicity in an oral OECD 422 study in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted on June 25, 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

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 or test system 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

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

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).

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.

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 started approximately 20 to 25 minutes post-dosing and lasted until approximately at 45 to 60 minutes post-dosing. 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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

According to the study director’s detailed data analysis, 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, however the difference between mid dose males and control animals consistently remained <10% and was thus not considered as toxicologically relevant. 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. However, differences in body weight gain between high dose males and control animals did not reach statistical significance at any time point after week 6, so this was not considered as a test item-related effect. In sum, 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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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. Moreover, values of these parameters of all treated females were within 95 percentile historical ranges (except one female of low dose), hence, effect was not related to test item treatment.
- Dose independent statistically significant increase was noted in haemoglobin in low and mid dose female, but values of these parameters of all treated females were within 95 percentile historical ranges (except one female of low dose), hence, 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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

Endocrine findings:

no effects observed

Description (incidence and severity):

Hormone Analysis: The study director concluded that thyroid hormone level (TSH, T3 and T4) of male and female rats of the treatment groups was comparable with that of the vehicle control group.
In more detail, serum T4 level was statistically significantly increased in female rats. However, serum T4 level was not statistically significantly altered in males, both serum TSH and T3 levels were unchanged and no changes were observed in thyroid weight or histopathology. In the absence of any correlating effect and considering the unchanged thyroid hormone levels in the OECD 414 and OECD 422 studies, this was not considered as a relevant test item-related effect.
Please also refer to attached Tables below.

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)
Please also refer to attached Tables below.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehavioural Observations:
Home Cage Observations:
- In the home cage, all rats from the treatment (G2, G3, and G4) and the vehicle control groups (G1) showed normal posture, 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 in home cage during NBO
Please also refer to attached Tables below.

Handling Observations:
NBO performed during handling of rats did not reveal any abnormalities. All rats revealed normal behaviour during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). No rat showed lacrimation, salivation, or piloerection. Eyelids were wide open in all rats. Eye and skin examination did not reveal any abnormality in any rat.

Open Field Observation:
In the open field, all rats from the treatment and vehicle control groups showed normal gait, mobility, arousal, and respiration during the ‘three-minute observation period’. Clonic and tonic movements, vocalisation, stereotypy, and bizarre behaviour were absent in this assessment. The rearing, urination, and defecation counts of male and female rats from the treatment groups were generally comparable with those of the vehicle control group with a few exceptions as follows:
- 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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: Hormone analysis

Critical effects observed:

no

Estrous Cycle Status of Termination

Estrous status of female rats on day of necropsy was as mentioned below in this table

Rat N°	Phase	Rat N°	Phase	Rat N°	Phase	Rat N°	Phase
11	D	31	D	51	E	71	P
12	E	32	E	52	E	72	E
13	P	33	D	53	D	73	D
14	P	34	D	54	P	74	E
15	D	35	E	55	D	75	-
16	P	36	E	56	D	76	M
17	P	37	P	57	E	77	E
18	E	38	D	58	D	78	P
19	E	39	E	59	D	79	D
20	P	40	E	60	E	80	D

  Key: E = Estrous, P = Proestrous, M = Metestrous, D = Diestrous and - = Not applicable

Please also refer to attached additional Tables below.

Conclusions:

- Based on above results, it is concluded that N-Butylaminoethanol did not produce any toxicity or test-item related 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 change was observed during the ophthalmological examination of the treated rats.

No treatment related changes were observed in the neurobehavioural observation and functionalobservational battery performed in rats from the reatment groups.

No treatment related changes were observed in the mean body weight in females and food consumption oof all treatment groups as compared to control. In males, body weight gain was slightly (<10 %) decreased in mid dose males from week 7 until week 13. Due to the marginal extend, this was not considered as toxicologically relevant. Occasional, unique statistically significant decreases in body weight gain observed in high dose males at weeks 2, 4 and 6, but not thereafter, were not considered as a consistent test-item-related effect.

No treatment related changes were observed in the heamatology, clinical chemistry and urinalysis parameters of the treatment groups as compared to control.

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 histopathology, up to dose level 240 mg/kg bw/day.

Based on above results, it is concluded that N-Butylaminoethanol did not produce any relevant toxicity or test-item related 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.

Reference 2

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 for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 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 or test system 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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
- Statistically significantly lower mean body weight (< 10 %) 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, furthermore changes remained below 5% compared to concurrent control animals.
- Statistically significant reductions in mean body weight gain in the high dose group during treatment days 1-8, 15-22, 22-29 and 1-50, clearly exceeding a reduction by 10% compared to control animals, 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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not reveal any test item related effect.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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).
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.
Please also refer to attached Tables below.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

240 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are repeated dose toxicity studies via the oral route of exposure available for N-Butylethanolamine.

An OECD 408 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 (10 rats/sex/group). The following dose levels have been set based on a 14 -day range finder study and an OECD 422 study conducted with N-butylethanolamine: G1 (vehicle control): 0 mg/kg bw/day; G2 (low dose): 60 mg/kg bw/day, G3 (mid dose):120 mg/kg bw/day andG4 (high dose): 240 mg/kg bw/day. 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.

In the 90 -day repeated dose toxicity study, 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 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 haematology, clinical chemistry and urinalysis parameters. Slight changes in body weight gain in mid dose males and occasionally occurring statistically significant decreased in body weight gain of high dose males after 2, 4 and 6 weeks, but not thereafter, were not considered as test item-related, toxicologically relevant adverse effect. 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.

In 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.

Moreover, there is an oral OECD 422 in rats available for N-Butylaminoethanol. 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). 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.

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

In the Low and Mid dose group rats no signs of toxicity on clinical signs, 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.

In the High dose group rats no signs of toxicity on mortality, NBO, FOB, organ weight, clinical pathology (absolute and relative) was observed. No treatment related macroscopic and microscopic lesions were observed. The 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. There were no effects on endocrine sensitive endpoints observed.

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.

The corresponding dose range finder covered a period of 2 weeks and was conducted to evaluate the potential toxicity of n-butylaminoethanol in Wistar rats following daily oral (gavage) administration for at least 14 consecutive days. The results was used for the safety assessment of the test item and to select dose levels for possible further long-term toxicity studies. This study was conducted in compliance with OECD Principles of GLP (1998). 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.

As result, 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.

In the low dose group 40 and the mid dose group 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) were observed. Furthermore, no abnormalities (external or internal) were found.

In the high dose group 400 mg/kg/bw/day, one mortality (male) on day 7 and two (male on day 13 and female on day 8) were noted. Furthermore, clinical signs like lethargy dyspnoea, abdominal breathing, gasping, rales piloerection, weakness, vocalisation and writhing in males (2 of 5) and females (3 of 5) were observed. 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.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
There is an oral 90 day study in rats for N-butylethanolamine (BEA) available. Moreover, there is an oral OECD 422 study (and the respective dose-range finder) for BEA available.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
There is no inhalation toxicity study in rats available for N-Butylaminoethanol. However, inhalation is the relevant route of exposure for this substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

Only one study via inhalation with a close analogue (Dibutylethanolamine) available.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

There is no dermal toxicity study available for N-Butylaminoethanol. However, an oral 90 day study in rats for N-butylethanolamine (BEA) is available. Moreover, there is an oral OECD 422 study (and the respective dose-range finder) for BEA available.

This is not the relevant route of exposure for this substance.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

There is no dermal toxicity study available for N-Butylaminoethanol. However, an oral 90 day study in rats for N-butylethanolamine (BEA) is available. Moreover, there is an oral OECD 422 study (and the respective dose-range finder) for BEA available.

This is not the relevant route of exposure for this substance.

Justification for classification or non-classification

N-butylethanolamine was found not to be toxic after oral exposure for 90 days to rats.

Based on these data, the substance does not need to be classified and labelled for STOT-RE according to Regulation (EC) No.1272/2008.