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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from a study report.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrabutylammonium bromide
EC Number:
216-699-2
EC Name:
Tetrabutylammonium bromide
Cas Number:
1643-19-2
Molecular formula:
C16H36N.Br
IUPAC Name:
tetrabutylazanium bromide
Test material form:
solid: crystalline
Remarks:
White to off white crystalline powder
Details on test material:
Name: Tetrabutylammonium bromide
CAS No: 1643-19-2
IUPAC Name: 1-Butanaminium, N,N,N-tributyl-, bromide (1:1)
Molecular Weight: 322.37 g/mol
Molecular Formula: C16-H36-N.Br
SMILES: [Br-].CCCC[N+](CCCC)(CCCC)CCCC

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animals were purchased from a CPCSEA approved vendor.
- Age at study initiation: 12 weeks
- Weight at study initiation:
G1: 221.98 to 278.92
G2: 224.82 to 284.80
G3: 209.92 to 284.61
G4: 219.54 to 284.03
- Fasting period before study:
- Housing: Rats were housed in standard polysulfone rat cages with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.
- Diet (e.g. ad libitum): Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier, was provided ad libitum to the rats.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 58 - 66 %
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES:
From: 30 July 2020
To: 02 September 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q® Water.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared fresh daily before dosing and used within the established stability period of 24 hours. Required quantities of the test chemical was weighed into pre-calibrated beakers for each dose level separately. The test chemical was dissolved with a small volume of vehicle (Milli-Q water) by stirring using a glass rod, and the volume was made up to the mark using a vehicle to get the final desired concentrations.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Milli-Q® water was used as a vehicle.
- Concentration in vehicle: Please find below-
Low Dose Group: 10 mg/mL (100 mg/kg bw)
Mid Dose Group: 20 mg/mL (200 mg/kg bw)
High Dose Group: 40 mg/mL (400 mg/kg bw)
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The method for determination of the test chemical concentration in dose formulation with the vehicle (Milli Q water) was successfully validated for specificity, linearity and range, intermediate precision, accuracy and precision at nominal concentration of 1 and 80 mg/mL. The test chemical was found to be stable in the vehicle at 1 and 80 mg/mL up to 24 hours for low dose and high dose, when stored at room temperature.

FORTIFICATION LEVELS
Method validation and stability test were carried out at the following concentrations in the vehicle (Milli-Q water.)
• Low Dose – 1 mg/mL (Nominal conc.)
• High Dose – 80 mg/mL (Nominal conc.)
The test chemical in the dose formulations was determined using High Performance Liquid Chromatograph (HPLC) with Mass Spectometer.
Chromatographic Analysis
Instrument: High performance liquid chromatography with mass spectrometer.
Column: Zorbax RX C-18, 250 mm long, 4.6 mm i.d., 5 µm particle size.
Cooler Temperature : 15°C
Mobile Phase : Pump A: 0.1 % (v/v) Formic acid in Milli-Q-Water
: Pump B: Acetonitrile
Mobile phase ratio : Pump A : Pump B (50:50 v/v)
Flow rate : 1.0 mL/min

MS conditions
Scan type: MRM
Ionization mode: ESI +ve (polarity)
Q1/Q3: 242.4/142
Q1/Q3: 242.4/99.8

Source parameters
Curtain gas: 10
CAD: 3
Ion spray voltage: 4200
Ion source gas (GS1): 50
Ion source gas (GS2): 60
Temperature: 500
De-clustering potential (DP): 60
Entrance potential (EP): 4.50
Collision energy (CE): 35
Collision cell exit potential (CXP): 5
• All the parameters were maintained constant throughout the analysis.
• The system suitability was checked on each day of analysis by the use of external working standard solutions, as detailed in system suitability test.
• Analyte peak in the sample was identified by comparing the retention time of analyte peak in the standard with that of analyte peak in the sample.
• Peak area of analyte for each injection were recorded.
• The chromatographic system was calibrated, by bracketing calibration standard injections before and after a set of sample injections, using working standard solutions
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused: Yes
- M/F ratio per cage: 1:1
- Length of cohabitation: Until the morning hours of the subsequent day.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data Available
- Further matings after two unsuccessful attempts: [no]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: No Data Available
Duration of treatment / exposure:
15 days (gestation Day 5 to 19)
Frequency of treatment:
Once Daily
Duration of test:
Up to gestation day 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (G1)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group (G2)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group (G3)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (G4)
No. of animals per sex per dose:
24 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based upon the available literature on the test chemical. In a previously done study according to OECD TG 422 (Source: Japan CHemical Collaborative Knowledge database or J-CHECK database; presented in this dossier under section 7.8.1 for toxicity to reproduction), two mortalities in high dose group (600 mg/kg bw) were observed. One male and One Pregnant female had died at high dose group. Thus, subsequent lower dose of 400 mg/kg bw was selected as a high dose.
- Rationale for animal assignment (if not random): GD 0 pregnant rats obtained on each day were randomly distributed to different groups by body weight stratification method using Provantis TM software. After grouping and ascertaining the group mean body weight, the rats were given accession number as applicable to the group on each day of assignment.
- Other: No Data Available

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once Daily during Acclimatization. Each rat was observed twice daily during treatment period for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment as there were no clinical signs of concern, the observation for morbidity and mortality was carried out once during weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs were performed twice a day, pre dose and post dose during treatment days and at least once on other days.

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20. The intermittent body weight gain was calculated for GD 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, and 17–20. Further the body weight gain for pre-treatment period (GD 0–5), treatment period (GD 5–19), and for entire gestation period (GD 0–20) was derived and analyzed only for rats pregnant at caesarean section. The corrected body weight was obtained by subtracting the unopened uterine weight from terminal body weight (body weight on GD 20). The corrected body weight gain was calculated by subtracting the body weight on GD 5 from the corrected body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, The quantity of food consumed by each female was calculated for the following intervals: gestation days GD 0–3, 3–5, 5–8, 8–11, 11–14, 14–17 and 17–20. Further food consumption for pre-treatment period (GD 0 - 5), treatment period (GD 5 - 19) and for entire gestation period (GD 0 - 20) was derived.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: No Data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Organs included in uterine examinations were examined. Also, thyroid gland was examined.

OTHER: No Data Available
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: No Data Available
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [all per litter]
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association. Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.
Historical control data:
Historical control data of the test facility was considering during data interpretation.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity at any dose level.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals survived to planned death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in maternal body weight or maternal body weight gain (corrected or uncorrected for uterine weight) were observed, with the exception of a significant increase in corrected body weight gain observed at 400 mg/kg/day. This effect was considered incidental as the body weights measured during different intervals of gestation period were unaffected by treatment and comparable to vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in food intake were observed, with the exception of two significant increases in food intake between GD 8 to 11 at 200 mg/kg (by 9% vs. controls) and at 400 mg/kg (by 10% vs controls). Both of these effects were considered incidental because the changes were marginal and because no significant changes in body weight were observed during the treatment period.
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:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes in thyroid hormone levels were observed. A mild, yet statistically significant, decrease in T4 levels was observed at 400 mg/kg, however this change did not show any associated inverse change in TSH values and the thyroid was normal on microscopic examination. Hence this change in T4, was considered incidental and therefore not toxicologically significant.
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):
Absolute weights and weights relative to body weight of gravid uterus, ovary, and thyroid gland remained comparable for pregnant females of control and treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological findings that could be attributed to the test item were observed at any dose level.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of the thyroid gland revealed no significant effects at any dose level.
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The number of pregnant animals were 23, 23, 24 and 24 at 0, 100, 200 and 400 mg/kg bw/day.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A biologically significant increase in post-implantation loss was observed at 400 mg/kg bw/day. Post implantation loss at 400 mg/kg (mean value, 1.7) was higher as compared to vehicle control (mean value, 0.78) and the historical control range (95% CI; 0.53 to 0.74). No significant changes in post implantation loss were observed at 100 or 200 mg/kg bw/day compared to the control group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant changes observed.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Bioloigcally relevant increaes in early and late resorptions were observed at 400 mg/kg bw/day compared to the control group.
Early resorptions at 400 mg/kg (mean value, 1.3) was higher as compared to vehicle control (mean value, 0.5) and the historical control range (95% CI; 0.44-0.62).
Late resorptions at 400 mg/kg (mean value, 0.5) was higher as compared to vehicle control (mean value, 0.3) and the historical control range (95% CI; 0.07- 0.14).
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed at any dose level.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant animals were 23, 23, 24 and 24 at 0, 100, 200 and 400 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
Gross evaluation of the placenta revealed no treatment-related effects.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
dead fetuses
early or late resorptions
maternal abnormalities
necropsy findings
number of abortions
pre and post implantation loss
total litter losses by resorption
Dose descriptor:
LOAEL
Remarks:
maternal developmental toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
early or late resorptions
pre and post implantation loss

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant changes observed.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live fetuses were 322, 318, 363, and 303 at 0, 100, 200 and 400 mg/kg bw/day, respectively. No dead fetuses were observed at any dose level.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No significant changes observed.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant changes observed.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed during external examinations of fetuses of rats treated up to 400 mg/kg/day. There was one incidence of a fetus with thread like tail [malformation] in the 100 mg/kg/day dose group (1/318 fetus) and one incidene of a small fetus [anomaly] in the 400 mg/kg/day dose group (1/302 fetus). These incidences were considered spontaneous in nature and not treatment-related
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Only one fetus in the study exhibited a skeletal malformation and that was a fetuses from the 100 mg/kg dose group that lacked the caudal vertebrae (centrum and arch absent). The same fetus had thread like tail [malformation] during the external examination. The skeletal malformation observed at 100 mg/kg/day was considered spontaneous in nature and not treatment-related. There were significant increases in skeletal variations of incomplete ossification of skull bones namely parietal, interparital, squmosal and hyoid bone at 100 and 200 mg/kg/day; a significant increase in delayed ossification of sternum no. 6 at 400 mg/kg/day; and significant increases in incomplete ossification of sternum no. 6 at 100 and 200 mg/kg/day. Delayed/incomplete ossification is generally considered transient changes and these finding denotes generalized growth delays which normally ossify late in gestation. They also do not have any general predictive value for teratogenicity [Carney and Kimmel (2007)]. Hence these variations were not considered treatment-related. Significant increases in skeletal anomalies of bilateral wavy ribs (4 to 12) were observed at 400 mg/kg/day as compared to vehicle control group. Wavy ribs are manifestations of delayed ossification, are transient and reversible and are not of developmental importance which are expected to resolve as the organism matures (Hayasaka et al., 1985; Kast, 1994; Mitchard & Stewart, 2014) and hence considered not treatment-treatment change.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed during visceral examinations at 100, 200 and 400 mg/kg/day. There was one incidence of moderate unilateral renal pelvis dilation [anomaly] in vehicle control group (1/157 fetuses), one incidence of heart innominate artery absent [anomaly] (1/144 fetuses) at 400 mg/kg/day; and another incidence of short innominate artery [anomaly] (1/144 fetuses) at 400 mg/kg/day. All of these incidences were considered spontaneous in nature and not treatment-related
Other effects:
no effects observed
Description (incidence and severity):
No significant changes in AGD were observed.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

    Summary of Maternal Survival, Pregnancy Status and Fetus Disposition

Parameters 

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

200

400

Total No. of rats found sperm positive / group

24

24

24

24

Duration of treatment 

GD 5 to 19 (total 15 days)

Caesarean section

(day of presumed gestation)

GD 20

Number of rats sacrificed at caesarean section

24

24

24

24

Number. of rats non-pregnant at caesarean section

1

1

0

0

Number of rats pregnant at caesarean section

23

23

24

24

Number of litters examined

23

23

24

24

Total number of fetuses

322

318

363

302

Total number of dead fetuses

0

0

0

0

 

 

 

 

 

Number of fetuses evaluated

 

 

 

 

a. External examination

322

318

363

302

b. Visceral examination

157

154

177

144

c. Skeletal examination

165

164

186

158

Summary of Maternal Data

 

Sex: Female

 

G10

mg/kg/day

G2 100

mg/kg/day

G3 200

mg/kg/day

G4 400

mg/kg/day

Group size

 

    N

23

23

24

24

Pregnant at C/S

 

   N

23

23

24

24

Gravid Uterus Weight

[a]

Mean SD

79.884

22.845

78.643

18.518

85.352

8.759

71.800

 

23.442

Number of

Corpora Lutea

[k]

Mean

SD

Sum

17.5

1.5

403.0

17.4

2.5

400.0

17.8

1.4

426.0

17.8

2.8

428.0

Number of

Implantation

[k]

Mean SD

Sum

14.8

3.9

340.0

15.0

3.2

344.0

16.1

1.7

386.0

14.3

4.2

343.0

Number of dams with Early Resorption

 

  N

10

15

18

16

Number of Early Resorptions

[k]

Mean SD

Sum

0.5

0.7

12.0

1.1

1.0

25.0

0.8

0.6

20.0

1.3

 

1.3

30.0

% Early Resorption

/Animal

[k]

Mean SD

3.47

4.40

8.64

11.10

5.03

3.40

9.69

11.65

Number of dams with Late Resorption

 

 N

6

1

3

11

Number of Late Resorptions

[k]

Mean SD

Sum

0.3

0.4

6.0

0.0*

0.2

1.0*

0.1

0.3

3.0

0.5

0.5

11.0

% Late Resorption

/Animal

[k]

Mean SD

2.40

4.89

0.27 *

1.30

0.90

2.49

3.11

3.62

Number of dams with

Resorptions

[f]

 N

13

15

19

19

Total Number of Resorptions (Early + Late)

[f]

Mean SD

Sum

0.8

0.8

18.0

1.1

1.0

26.0

1.0

0.6

23.0

1.7

1.4

41.0

Pre-implantation Loss/Animal

[k]

Mean SD

2.74

3.32

2.43

3.13

1.67

1.40

3.54

3.97

% Pre-implantation

Loss

[k]

Mean

SD

16.2

21.0

13.4

17.6

9.3

8.0

20.2

23.5

Post-implantation Loss/Animal

[k]

Mean SD

0.78

0.80

1.13

1.01

0.96

0.62

1.71

1.43

% Post-implantation

Loss (%)

[k]

Mean

SD

5.9

6.5

8.9

11.1

5.9

3.9

12.8

11.9

Note: Gross evaluation of placenta revealed no findings

[a] - Anova & Dunnett(Log)

[k] - Kruskal-Wallis & Wilcoxon; * = p < 0.05

[f] - Chi-Squared & Fisher's Exact

 Summary of Litter Data

 

Sex: Female

 

G10

mg/kg/day

G2 100

mg/kg/day

G3 200

mg/kg/day

G4 400

mg/kg/day

Total Number of fetuses

 

Sum

322

318

363

302

Total Number of Dead Fetuses

 

Sum

0

0

0

0

Total Number of Live fetuses

 

Sum

322

318

363

302

Live Male fetus

 

Sum

178

161

173

141

% Male Fetus

 

 

55.3

50.6

47.7

46.7

Mean Fetal

Weight- Male

(g)

[c]

Mean SD

3.707

0.259

3.772

0.216

3.786

0.234

3.736

0.263

Mean AGD- Male (mm)

[c]

Mean

SD

2.65

0.11

2.69

0.13

2.66

0.11

2.63

0.08

Live Female fetus

 

Sum

144

157

190

161

% Female Fetus

 

 

44.7

49.4

52.3

53.3

Mean Fetal

Weight- Female

(g)

[c1]

Mean SD

3.500

0.321

3.601

0.204

3.582

0.228

3.530

0.235

Mean AGD- Female (mm)

[c1]

Mean

SD

0.90

0.08

0.89

0.07

0.92

0.14

0.90

0.06

Mean Fetal

Weight -Male+Female

(g)

[c2]

Mean SD

3.605

0.310

3.689

0.205

3.682

0.233

3.606

0.246

Mean AGD

Male + Female (mm)

[c2]

Mean

SD

1.84

0.28

1.81

0.29

1.75

0.29

1.67

0.31

 

 

Applicant's summary and conclusion

Conclusions:
In this OECD 414 study conducted with tetra butyl ammonium bromide (CAS 1643-19-2) in Wistar rats, NOAEL values for maternal systemic toxicity, maternal developmental toxicity and fetal developmental toxicity were considered at 400, 200 and 400 mg/kg bw/day, respectively.
Executive summary:

A study according to Prenatal Developmental Toxicity Study, adopted: 25th June 2018 was performed using a total of 96 pregnant female Wistar rats. The test chemical was administered to these pregnant female Wistar rats by oral: gavage route and the vehicle used to dissolve the test chemical was Milli-Q water. The test chemical was soluble in distilled water in the in-house solubility test and therefore, Milli-Q water was chosen as the vehicle. The 96 pregnant females were randomized into 4 experimental groups containing 24 animals each. The groups, viz., G1, G2, G3 and G4 received 0, 100, 200 and 400 mg/kg bw/day of the test chemical, respectively. The test chemical was administered to these pregnant rats during Gestation Day 5 to 19 at the frequency of once daily. The doses were selected based upon the available literature on the test chemical. In a previously done study according to OECD TG 422 (Source: Japan CHemical Collaborative Knowledge database or J-CHECK database), two mortalities in high dose group (600 mg/kg bw) were observed. One male and One Pregnant female had died at high dose group. Thus, subsequent lower dose of 400 mg/kg bw was selected as a high dose. The following parameters and endpoints were evaluated in this study: maternal parameters (mortality, clinical signs, body weight, body weight gain, food consumption, gross pathology, gravid uterine weight, number of corpora lutea,implantation sites, early and late resorptions, pre and post implantation loss,implantation index) and fetal parameters (total number of live fetuses, total number of dead fetuses, sex ratio, fetal weight, anogenital distance, and external, visceral and skeletal observations). Approximately half the number of the fetuses from each litter were examined for visceral malformations and variations and the remaining half were evaluated for skeletal malformations and variations. In addition, from each dam, the thyroid was weighed and subjected to microscopic evaluation. Thyroid hormones levels were determined from the blood samples collected at terminal sacrifie (on GD 20).

Main findings from the study are summarized below:

• Mortality, clinical signs, and gross necropsy changes: There were no unscheduled deaths, clinical signs, or any gross pathological findings that could be attributed to the test item.

• Body weight and food intake: No toxiclogically significant changes in maternal body weight or maternal food intake were observed at any dose level.

• Thyroid hormone levels (T3, T4 and TSH), thyroid weight, and thyroid histology: No toxicologically significant changes in thyroid hormone levels were observed. A mild, yet statistically significant, decrease in T4 levels was observed at 400 mg/kg, however this change did not show any associated inverse change in TSH values and the thyroid was normal on microscopic examination. Hence this change in T4, was considered incidental and therefore not toxicologically significant.

• Maternal developmental parameters: No significant changes in any maternal developmental toxicity endpoint were observed at 100 or 200 mg/kg/day. Increases in early and late resorptions were observed at 400 mg/kg/day, and consequently post-implantation losses were higher at 400 mg/kg/day compared to the vehicle control group. The increases in early resorptions, late resorptions, and post-implantation losses at 400 mg/kg were all above the respective historical control range of the test facility. These increases were therefore considered to be biologically significant. No gross pathological changes in the placenta were observed at any dose level.

• Litter Parameters: The total number of fetuses were 322, 318, 363, and 303 at 0, 100, 200 and 400 mg/kg/day, respectively. No dead fetuses were observed at any dose level. No significant changes in litter size, sex ratio, AGD, or fetal weight were observed at any dose level.

• Fetal examination: The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item.

Based on the above findings, under the test conditions used in this study, the following NOAEL values were derived:

• NOAEL for maternal systemic toxicity was considered at 400 mg/kg/day since no evidence of systemic toxicity was observed at any dose level 100, 200 or 400 mg/kg/day.

• NOAEL for maternal developmental toxicity was considered at 200 mg/kg/day. Biologically significant increases in early resorptions, late resorptions, and post-implantation losses were observed at 400 mg/kg/day and this dose level was therefore considered as LOAEL for maternal developmental toxicity.

• NOAEL for fetal developmental toxicity was considered at 400 mg/kg/day since no evidence of fetal toxicity was observed at any dose level.