Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-05-06 - 2020-03-20 (experimental phase: 2019-06-20 - 2019-10-24)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Remarks:
Dose-range finding study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
Dose-range finding study
Adequacy of study:
supporting study
Study period:
2019-02-11 - 2019-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to the following guidelines with modifications (as this study is dose range finding)
Reason / purpose:
reference to other study
Remarks:
main study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted by the Council on June 25, 2018
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan: WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 11 to 13 weeks weeks (initiation of the acclimatisation)
- Weight at study initiation: mean: 242.5 ± 17.64 (SD), body weight range 214.6 - 274.0 g (prior to acclimatisation)
- Fasting period before study: No
- Housing: Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male plus one female). Mated female rats were housed individually. During the study, rats were housed in solid floor polypropylene rat cages (size: 41cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5/6 tier racks. The bottom of the cages was layered with clean sterilised rice husk as the bedding. Cages with bedding material and water bottles were changed twice a week.
- Diet (e.g. ad libitum): The rats were fed ad libitum with standard rodent pelleted diet (Certified Teklad Global 16% Protein Rodent Diet, Batch No 2016SC-090618MA, procured from Envigo, Inc., USA)
- Water (e.g. ad libitum): Unlimited supply of drinking water in polypropylene bottles (capacity 250 mL). Drinking water was filtered through Reverse Osmosis water filtration system.
- Acclimation period: Rats were acclimated in the experimental room for a period of 6 days prior to the cohabitation. During the acclimatisation period, rats were checked once daily for clinical signs of toxicity that would preclude use on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 58-66
- Air changes (per hr): 21 (March), 19 (April)
- Photoperiod (hrs dark / hrs light): 12 hours of lighting and 12 hours of darkness
- Mean light intensity (in LUX): 202 (March), 239 (April)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item was formulated in the vehicle at the appropriate concentration for each dose group. The dose formulations were prepared on each day prior to the dosing and thoroughly mixed using magnetic stirrer in order to maintain a homogeneous preparation during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Reverse osmosis (RO) water was selected as a vehicle based on solubility check.
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- test item in vehicle was stable up to 8 days.
- Concentration and homogeneity of dose formulations were analysed using a validated analytical method
- Two sets of samples (13 samples per set) were collected by sampling three aliquots (top, middle and bottom layers) from each concentration except the vehicle control (only one aliquot from middle layer).
- Sampling was done before the start of treatment and once during the treatment period for dose formulation analyses. One set of samples was used for analyses and other set of samples was stored refrigerated. The second set of samples was disposed of upon 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 are ± 10 % from nominal value and % CV < 10.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 per cage
- Length of cohabitation: not specified (until the requisite numbers of mated females were obtained)
- Proof of pregnancy: by vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From GD 5 to GD 19
Frequency of treatment:
once daily
Duration of test:
From GD 0 to GD 20
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)
Dose / conc.:
320 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8 females/ dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the result of a 14-day dose range finding study and in consultation with the study sponsor. In 14-day dose range finding study, at high dose, i.e., 400 mg/kg bw/day, 3 mortalities occurred with antemortem clinical signs of toxicity noted. At the mid and low doses, i.e., 125 and 40 mg/kg bw/day, respectively, no signs of toxicity was observed. Hence, following dose levels: low dose 60, mid dose - I 120, mid dose - II 240, and high dose 320 mg/kg bw/day were selected for the present study.

- Rationale for animal assignment (if not random): Mated females were assigned to dose groups by stratified randomisation on the basis of GD 0 body weights. Body weights of GD 0 were arranged in descending order, from the heaviest to the lightest on the first GD 0 date, and assigned in the same order on the subsequent days. Beginning with the heaviest weight, one rat was randomly assigned to each stratified group. In the event that the total number of rats mated on a given day was not an even multiple of the number of treatment groups, the mated females were assigned to complete the last incomplete stratification group and then assigned to the next stratification group until all GD 0 females for that day were assigned.

- Other:
Justification of Test System: The albino rat was selected as a test system because it has historically been shown to be a suitable model for prenatal developmental toxicity studies. This species is also recommended by the OECD and other regulatory authorities.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for mortality and morbidity.

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

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

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 10
- Organs examined: The non-gravid uteri were further examined by staining with 5 % ammonium sulphide to confirm non-pregnant status. Ovaries and gravid uteri including the cervix were removed and examined immediately. Ovaries, foetuses, and non-gravid uteri were preserved and identified.

OTHER: weight of the thyroid gland was recorded from all pregnant rats and preserved for possible histopathology. As histopathology was not performed, preserved thyroid gland discarded upon finalisation of the report.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including the cervix weight)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead foetuses
Fetal examinations:
- External examinations: Yes
A total of 58, 62, 65, 88, and 65 foetuses were examined in 0, 60, 120, 240, and 320 mg/kg bw/day dose groups, respectively.

Other:
- Number of male foetuses
- Number of female foetuses
- Body weight of male foetuses
- Body weight of female foetuses
- Foetus sex (based on ano-genital distance)
- Ano-genital distance
Statistics:
- All raw data was processed to determine group means and standard deviations with statistical significance between the control and treatment groups assessed using validated statistical software. The data were summarised in tabular form. Non-pregnant rats were excluded from statistical analysis. Found dead rats were considered for statistical analysis of number of corpora lutea, number of implantation sites and pre-implantation loss.

- The parametric data (body weight, body weight change, food consumption, and organ weight) were subjected to Barlett'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 homogeneity of variance, statistical analysis was extended following a decision tree.

- The non-parametric data (mortality rate, pregnancy rate etc.) were analysed using the Chi-square test.

- Count data {viz., foetal count, number of corpora lutea, number of implants, number of live foetuses, number of dead foetuses} were subjected to non-parametric Kruskal-Wallis test.

- AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.

- Flags of significant difference between control and treated groups (single arrow for p greater equal 0.05 and double arrowed for p greater equal 0.01) were given in the table along with the footnote.
Historical control data:
JRF has historical control data on the background incidence of foetal malformations and variations in the Wistar rats used for this study
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Salivation was observed approximately 10 to 15 minutes after dosing in 7 of 8 rats and persisted for approximately 45 to 50 minutes. This finding is considered a response to the dose solution and toxicologically irrelevant.
- Piloerection and weakness were noted for both rats that succumbed to treatment as well as one surviving rat and are considered test item related.
- No clinical signs of toxicity were observed up to a dose level of 240 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
- Two mortalities were observed in 320 mg/kg bw/day dose group both on gestation day 13. Both rats were found dead with antemortem signs of weakness and piloerection. These mortalities are considered treatment related.
- No mortality was observed in any other group during the dosing period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight, body weight change, and 20th day corrected body weight of the pregnant female rats were comparable to control throughout the dosing period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- The mean food consumption of the pregnant female rats was comparable to control with the exception of statistically significantly decreased in food consumption during gestation period days 5-8 and 8-11 for the 120 and 320 mg/kg bw dose groups, respectively.
- Due to recovery of the effect and in the absence of other supporting findings in mean body weight and body weight change, decreased food consumption in 120 and 320 mg/kg bw dose groups were considered incidental and non-adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute and relative weight of thyroid gland was comparable between test item treated groups and the control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Internal (gross) examination of the found dead and terminally sacrificed female rats did not reveal any lesion of pathological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease in post-implantation loss and increase in mean percent of live foetuses were observed in 240 mg/kg bw dose group. These changes were due to increase in number of live foetuses as compared to control group and have no toxicological significance.
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Description (incidence and severity):
Comparable to control for all test groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
Mean percent of live foetuses comparable to control group
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate was 75.0 % in the control group and 87.5 % for each of the 60, 120, 240, and 320 mg/kg bw/day dose groups.
Other effects:
no effects observed
Description (incidence and severity):
Implantation sites, relative uterine weight of the pregnant female rats, the mean numbers of corpora lutea were comparable to control for all test groups.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male foetus body weights were statistically significantly decreased when compared to control. The body weights for female foetuses and for a composite of foetuses of both the sexes were lower than control (8.81 % and 10.00 %, respectively) without statistical significance. The lower foetus body weights were also lower than the historical control data range (3.54 for male foetuses, 3.39 for female foetuses, and 3.47 for composite of male and female foetuses) and were not correlated with maternal body weight. Hence the foetal body weight findings were considered as treatment related.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean foetal count of male, female, and total foetuses (male + female) were comparable to control.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Male sex ratio and male to female sex ratio was comparable to control.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External examination of the foetuses showed no increased incidence of malformation or variations for foetuses from the treated groups as compared to control.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The mean anogenital distance of male, female, and total foetuses (male + female) were comparable to control.
Conclusions:
On the basis of results obtained in the present study following dose levels are suggested for the definitive prenatal developmental oral toxicity study of N-butylaminoethanol:
Option 1: 60 (low), 120 (middle), 240 (high) md/kg bw/day
Option 2: 65 (low), 130 (middle), 260 (high) md/kg bw/day
Option 3: 70 (low), 140 (middle), 280 (high) md/kg bw/day
Executive summary:

Methods: This study was performed to evaluate the dose levels for a definitive prenatal developmental oral toxicity study of N-butylaminoethanol in Wistar rats. N-butylaminoethanol was administered daily from gestation day (GD) 5 to 19, by gavage, to 8 mated female rats per group at dose levels of 60, 120, 240, and 320 mg/kg bw/day. The control group received reverse osmosis water only on the same regimen as the test item groups. Rats were observed twice daily, for mortality, morbidity, and clinical signs of toxicity. Maternal body weight and food consumption were recorded throughout the gestation period. All surviving female rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed and examined for the numbers of implantation sites, early and late resorptions and numbers of live and dead foetuses. The ovaries were excised and the number of corpora lutea counted. The foetuses were identified for sex, weighed and examined for external abnormalities. The anogenital distance was measured for all foetuses. Dose formulations were analysed for test item concentration and homogeneity prior to initiation of treatment and once during the treatment period.

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

N-butylaminoethanol at 60, 120, and 240 mg/kg bw/day:

- no treatment related effects observed

N-butylaminoethanol at 320 mg/kg bw/day:

- A total of two rats were found dead. These rats exhibited piloerection and weakness before death and are considered treatment related.

- Treatment related clinical signs included salivation, piloerection, and weakness. Salivation was observed approximately 10 to 15 minutes after dosing in 7 of 8 rats and persisted for approximately 45 to 50 minutes. This finding is considered a response to the dose solution and toxicologically irrelevant. Piloerection and weakness were noted for both rats that succumbed to treatment as well as one surviving rat and are considered test item related.

- The mean body weights, body weight changes, food consumption, and 20th day corrected body weight for the treated pregnant female rats were comparable to control.

- The mean absolute and relative uterine weights of the pregnant female rats were comparable to control.

- External and internal (gross) examination of the found dead and terminally sacrificed female rats did not reveal any lesions of pathological significance.

- The absolute and relative weights of thyroid gland were comparable to control.

- No differences from control were noted for the mean numbers of corpora lutea, implantation sites, resorptions (early and late), mean percent of live foetuses, dead foetuses, preimplantation loss, and post-implantation loss.

- The mean foetal count of male, female, and total foetuses (male + female) was comparable to control.

- Male foetus body weights were statistically significantly decreased when compared to control. The body weights for female foetuses and for a composite of foetuses of both the sexes were lower than control (8.81 % and 10.00 %, respectively) without statistical significance.

- The lower foetus body weights were also lower than the historical control data range (3.54 for male foetuses, 3.39 for female foetuses, and 3.47 for composite of male and female foetuses) and were not correlated with maternal body weight. Hence, the foetal body weight findings were considered as treatment related.

- The mean anogenital distance of male, female, and total foetuses (male + female) were comparable to control.

- Male sex ratio and male to female sex ratio was comparable to control.

- No incidence of malformation/variation was observed during the external examination of foetuses.

Conclusion: On the basis of results obtained in the present study following dose levels are suggested for the definitive prenatal developmental oral toxicity study of N-butylaminoethanol:

Option 1: 60 (low), 120 (middle), 240 (high) md/kg bw/day

Option 2: 65 (low), 130 (middle), 260 (high) md/kg bw/day

Option 3: 70 (low), 140 (middle), 280 (high) md/kg bw/day

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 414 (Prenatal Developmental Toxicity Study)
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 test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 12 to 13 weeks (initiation of the acclimatisation)
- Weight at study initiation: mean: 223.16 ± 12.19 (SD), body weight range 194.6 – 256.7g (prior to acclimatisation)
- Fasting period before study: No
- Housing: Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male plus one female). Mated female rats were housed individually. During the study, rats were housed in solid floor polypropylene rat cages (size: 41cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless-steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5/6 tier racks. The bottom of the cages was layered with clean sterilised rice husk as the bedding. Cages with bedding material and water bottles were changed twice a week.Chemical and microbial contaminant analysis of samples of the bedding and enrichment materials are being checked at every six months intervals.
- Diet (e.g. ad libitum): The rats were fed ad libitum with standard rodent pelleted diet (Certified Teklad Global 16 % Protein Rodent Diet, Batch No 2016SC-010419MA, procured from Envigo, Inc., USA). Received Food: with certificate of analysis of nutrient content from the food supplier. Quality of food was monitored incompliance with JRF/TOX/SOP-2077. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Unlimited supply of drinking water in polypropylene bottles (capacity 250 mL). Drinking water was filtered through Reverse Osmosis water filtration system. Quality of water was monitored in compliance with JRF/TOX/SOP-2077. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: A total of 100 male and 120 female rats were obtained to permit the selection and/or replacement of individuals before the start of the cohabitation and acclimated in the experimental room for a period of 6 days prior to the cohabitation. During the acclimatisation period, rats were checked once daily for clinical signs of toxicity that would preclude use on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 66-68
- Air changes (per hr): 17 (June) or 16 (July)
- Photoperiod (hrs dark / hrs light): 12 hours of lighting and 12 hours of darkness
- Mean light intensity (in LUX): 207 (June), 241 (July)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantity of the test item for each dose group was received from the Test Item Control Office (TICO). Test item was formulated in the vehicle. The dose formulations were prepared every day prior to the dosing and thoroughly mixed using magnetic stirrer in order to make a homogeneous preparation.

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

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

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Rats were observed daily, twice, for mortality and morbidity.

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

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

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

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

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

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

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

The remaining 50 % of the foetuses from each litter were subjected to the detailed skeletal evaluation after processing and staining with alizarin red. The skeletal evaluation was performed, as per JRF/TOX/SOP-232, according to Staple’s and Schnell method. The foetuses were preserved in isopropyl alcohol and glycerin solution.
Statistics:
- Data pertaining to individual rats were provided.
- All raw data were processed to determine group means and standard deviations with statistical significance between the control and treatment groups using validated statistical software.
- The parametric data (body weight, body weight change, food consumption, hormones (T3, T4, and TSH) results, and organ weight) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data do not meet the homogeneity of variance, statistical analysis was extended following a decision tree.
- The non-parametric data (mortality rate, pregnancy rate, foetal observations etc.) were analysed using Chi-square test.
- Count data (viz., foetal count, number of corpora lutea, number of implants, number of live foetuses, number of dead foetuses, number of early and late resorption) were subjected to non-parametric Kruskal-Wallis test.
- AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
- Non-pregnant rats were not subjected to statistical analysis.
- Flags for significant difference between control and treated groups (single arrow for p ≤ 0.05 and double arrows for p ≤ 0.01) were given in the table along with the footnote.
Historical control data:
JRF has historical control data on the background incidence of foetal malformations and variations in the Wistar rats.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical sign of toxicity was observed up to a dose level of 140 mg/kg bw/day.
In 280 mg/kg bw/day dose group, salivation was observed approximately 10 to 15 minutes after dosing on a few days in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding was considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and body weight changes of the pregnant female rats were comparable between the control and the test item treated groups up to a dose level of 140 mg/kg bw/day.

For the 280 mg/kg bw/day dose group, a statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. A statistically significant decrease in day 20 corrected body weight was also noted for this group. In addition, statistically significant decreases in mean body weight change were observed during the gestation days 5-8, 8-11, 17-20, and 5-20. Decreased mean body weight change for this group were also observed during the gestation period days 11-14 and 14-17 without statistical significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the pregnant female rats was comparable between the control and the test item treated groups up to a dose level of 140 mg/kg bw/day.
Statistically significant decreased mean food consumption was seen for 280 mg/kg bw/day dose group for gestation days 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.
Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test item treatment did not lead to any alteration in the absolute and relative organ weight of the thyroid gland.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Internal (gross) examination of the terminally sacrificed control and test item treated female rats did not reveal any lesion of pathological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination of the thyroid gland revealed ultimobranchial cyst/s in 2 and 1 female rats of the control and 280 mg/kg bw/day dose group, respectively, and considered as spontaneous or incidental and therefore non-adverse in nature.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Thyroid Hormone Analysis: Serum level of T3, T4, and TSH in female rats belonging to test item groups was comparable to the control group.
- External a examination of the terminally sacrificed control and test item treated female rats did not reveal any lesion of pathological significance.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
280 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (actual dose received)
Basis for effect level:
other: All examined parameters

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean weight of male, female and total foetuses (male + female) was comparable between the control and the test item treated groups up to the dose level of 140 mg/kg bw/day.
In the 280 mg/kg bw/day dose group, male foetus body weight was statistically significantly decreased (- 6.57 % than control group) when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetal body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male sex ratio were comparable between the control and the test item treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean count of male, female and total foetuses (male + female) was comparable between the control and the test item treated groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
A total of 278, 264, 272, and 252 foetuses were examined in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
No external anomalies were observed in foetuses of the control and treatment groups up to the dose level of 280 mg/kg bw/day except one runt foetus (1/14) in the 280 mg/kg bwt/day dose group. This finding was comparable with that of the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 143, 137, 143, and 131 foetuses were observed for the skeletal evaluation in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
There were several incidences of the statistically significant differences in the skeletal variations in all test item treated groups (see Table 1, "Any other information on results"). These decreased skeletal variations were only because of higher number of incidences in control group compared to treatment and have no toxicological relevance. Due to the lack of dose dependency, the increase in number of foetuses and litters with Rib: Full supernumerary was considered as incidental and non-adverse (see Table 2, "Any other information on results"). In case of xiphisternum, the data indicate a delayed ossification associated with reduced foetal weights i.e., a delay in foetal development rather than a direct effect on bone tissue. In addition, this increase in the number of foetuses with Xiphisternum: Unossified was within the range of historical control data therefore considered as non-adverse in nature.
Visceral malformations:
no effects observed
Description (incidence and severity):
A total of 135, 127, 129, and 121 foetuses were examined in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
No gross treatment related visceral findings were observed in foetuses of the control and the treatment groups up to the dose level of 280 mg/kg bw/day with the exception of dilated ureter seen in one foetus from the control group. There were no any other visceral findings in any dose groups.
Other effects:
no effects observed
Description (incidence and severity):
The mean anogenital distance of male and female foetuses were comparable between the control and the test item treated groups

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
280 mg/kg bw/day (actual dose received)
Basis for effect level:
other: all examined parameters

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
280 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

Table 1: Statistically significant decrease in skeletal variations

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

Table 2: Statistically significant increase in skeletal variations

 Group  G1  G2  G3  G4
 Dose (mg/kg bw/day)  0 70   140  280
 Total N° of Foetuses Examined  143  137  143 131 
Rib: Full supernumerary 1 3 10 (increase) 0
Xiphisternum: Unossified 0 5 (increase)  8 (increase)  13 (increase)
Total N° of Litters Examined  23  23  22  22
 Rib: Full supernumerary  1  3   6 (increase)  0

Applicant's summary and conclusion

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

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

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

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

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

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

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

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

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

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

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

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