Tetrabutylammonium bromide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

other: authoritative database

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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

Details on test material:

- Name of test material (as cited in study report):Tetrabutylammonium bromide
- Molecular formula :C16H36BrN
- Molecular weight : 322.37 g/mole
- Substance type:Organic
- Physical state:Solid
- Purity was reported to be 100.0%

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Details on species / strain selection:

The rats were 10 weeks of age when dosing was initiated.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Atsugi breeding center.
- Age at study initiation: (P) 10 wks;
- Weight at study initiation: (P): Male 389 to 452 g Female 219 to 266 g
- Fasting period before study: No data available
- Housing: Animals were housed one per by sex in a metal net cage, during the mating period two per sex per cage will mother were housed individually in plastic cages.
- Diet (e.g. ad libitum): solid feed (NMF, Oriental Yeast Co., Ltd.), ad libitum.
- Water (e.g. ad libitum): Drinking water (NMF, Oriental Yeast Co., Ltd.), ad libitum.
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 ℃
- Humidity (%): 50 ± 20 %
- Air changes (per hr): 10-15 times per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water

Details on exposure:

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test chemical dissolved in Water for injection

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle:0, 60, 180, 600 mg/kg/day, Recovery 0, 600 mg/kg/day (R600)
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: Overnight
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further mating after two unsuccessful attempts: [no / yes (explain)] No data available
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Duration of treatment / exposure:

Main study:
Male : 42 days
Female : 41-53 days (from 14 days before mating to day 4 of lactation)

Recovery groups:
Male and females: 42 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control Group (G1)

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day

Remarks:

Low Dose Group (G2)

Doses / concentrations 3

Dose / conc.:

180 mg/kg bw/day

Remarks:

Mid Dose Group (G3)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day

Remarks:

High Dose Group (G4)

No. of animals per sex per dose:

Total: 116 animals
Test group
0 mg/kg bw/day:12 male and 12 female
60mg/kg bw/day:12 male and 12 female
180mg/kg bw/day:12 male and 12 female
600mg/kg bw/day:12 male and 12 female

Recovery group
0 mg/kg bw/day:5 male and 5 female
600 mg/kg bw/day:5 male and 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

Rats in the recovery groups were allowed to recover for 14 days after the final dose.

Positive control:

Not included

Examinations

Parental animals: Observations and examinations:

General toxicity: Mortality, clinical signs, detailed clinical signs, body weight (twice per week), food intake (twice per week), sensory reactivity (auditory response, approach response, touch response, tail pinch response, pupillary reflex, aerial righting reflex, landing foot splay), functionality (grip strength, motor activity), urinalysis (pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen, color, urine sediment, crystallization urin volume, osmolality), hematology (RBC, Hb, Ht, MCV, MCH, MCHC, reticulocytes, platelets, PT, APTT, FIB, WBC, Lymphocytes, neutrophils, eosinophils, basophils, monocytes, leucocytes), clinical chemistry (AST, ALT, LDH, GTP, ALP, total cholesterol, TG, PL, total bilirubin, glucose, BUN, creatinine, Na, K, Cl, Ca, P, Total protein, albumin, A/G).
Reproductive endpoints: estrous cyclicity, days until copulation, copulation index, insemination index, fertility index, number of pregnant animals, number of female with live pups, delivery index, gestation period, number of corpora lutea, number of implantation sites, implantation index, number of stillborn pups, number of liveborn pups, number of stillborn pups, live birth index, pup viability, sex ratio, pup body weight, gross findings in pups.

Oestrous cyclicity (parental animals):

Count of estrous, mean duration of estrous, index of animals with abnormal estrous.

Sperm parameters (parental animals):

No data available

Litter observations:

Number of live pups, number of dead pups, gross findings, pup weight, and sex ratio.

Postmortem examinations (parental animals):

Organ weight (brain thyroid, thymus, heart, liver, spleen, kidney, adrenal, testis, epididymides), gross findings and histolopathology (adrenal, bone and bone marrow, cerebellum, cerebrum, epididymides, heart, intestine [duodenum, jejunum, ileum, cecum, colon, rectum], kidney, liver, lung, lymph nodes, ovary, parathyroid, pituitary, prostate, sciatic nerve, seminal vesicle, spinal cord, spleen, stomach, testis, thymus, thyroid, urniary bladder, uterus). The number of corpora lutea and implantation sites were also recorded.

Postmortem examinations (offspring):

Gross examinations were carried out.

Statistics:

Fisher test, Dunnett test, and Bartlett method firstly test was applied.

Reproductive indices:

Copulation index, Fertility index, Gestation index, Delivery index and Live birth index.

Offspring viability indices:

Viability (survival rate) on Day 4 after birth was recorded.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: Red urine (600 mg/kg)
Females: None

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Deaths occured in 1 male and 1 pregnant female at 600 mg/kg. Before death, the male showed fracture of incisors, soft stool and a decrease in the amount of feces and the female showed staining of lower abdominal fur and staggering gait.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Decreased body weight gain at 600 mg/kg
Females: Increased body weight gain at 600 mg/kg, 1-15 day. Decreased body weight gain in recovery group at 600 mg/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: Increased food consumption at 600 mg/kg
Females: Increased food consumption at 180 and 600 mg/kg

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In blood chemistry examination, males in the 600 mg/kg group showed a high value in AST and a low value in blood urea nitrogen and females showed high values in AST and LDH and low values in blood urea nitrogen and creatinine. The females in the 600 mg/kg group showed a high value in calcium as well. These changes were not observed after the end of the recovery period showing reversibility of the changes.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males in the 600 mg/kg group showed red urine sporadically and light brown urine was observed at urinalysis, but red urine was a transient change.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males: Hypertrophy of perilobular hepatocytes (600 mg/kg), Diffuse hyperplasia in mucosa in the cecum (180 and 600 mg/kg), Cell infiltration in mucosa in the cecum, colon and rectum (Recovery, 600 mg/kg)
Females: Hypertrophy of perilobular hepatocytes (600 mg/kg), Diffuse hyperplasia in mucosa in the cecum (180 and 600 mg/kg), Cell debris in crypt in the rectum (600 mg/kg), Cell infiltration in mucosa in the cecum, colon and rectum (Recovery, 600 mg/kg), Hypertrophy of perilobular hepatocytes, erosion in the duodenum, atrophy of lymphoid follicle in the spleen, erosion in the glandular stomach, atorophy of thymus (600 mg/kg, dead animal)

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

In the 600 mg/kg group, the number of females that showed abnormalities in estrous cycle tended to be high.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

In parent animals in the 60 and 180 mg/kg groups, there were no test article-related changes in the number of days until copulation, copulation index, insemination index or fertility index. There were no test article-related changes in the delivery index, length of gestation period, number of corporalutea, number of implantation sites, implantation index, number of live born pups or sex ratio, and there were no test article-related changes in parturition condition or lactation observations.
In the 600 mg/kg group, the number of females that showed abnormalities in estrous cycle tended to be high, the number of days until copulation tended to be high, copulation index, insemination index or fertility index tended to be low values, a high value in stillborn index, and low values in viability of pups (day 0 and day 4 of lactation)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

180 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

2

Dose descriptor:

LOAEL

Remarks:

General toxicity

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

3

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

180 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

4

Dose descriptor:

LOAEL

Remarks:

Reproductive toxicity

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Low viability (LD 0-4) was observed at 600 mg/kg (86.7%) compared to control group (97.8%).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased pup body weights were observed on LD 0 at 600 mg/kg (male mean, 5.9 g; female mean, 5.8 g) compared to controls (male mean, 6.8 g; female mean, 6.4 g) and on LD 4 at 600 mg/kg (male mean, 8.2; female mean 7.9 g) compared to controls (male mean 10.2 g; female mean, 9.6 g)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test chemical-related changes in external observation or necropsy on day 4 after birth

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios at birth (Total numnber of males / total number of pups) were 0.50, 0.58, 0.46, and 0.51 at 0, 60, 180 and 600 mg/kg, respectively.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

180 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

gross pathology

other: sex ratio

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Any other information on results incl. tables

Fertility and pregnancy data in rats treated orally with Tetrabutylamrnonium bromide in the combined repeat dose and reproductive/developmental toxicity screening test
	0 mg/kg	60 mg/kg	180 mg/kg	600 mg/kg
Number of pairs examined	12	12	12	12
Estrouscycle	4.1	4.2	4.2	4.4
Irregular estrous cycle	0/12	1/12	0/12	4/12
Number of pairs with successful mating	12	12	12	12
Copulationindex (%) a)	100.0	100.0	100.0	83.3
Number of pregnant females	12	11	11	8
fertilityindex (%) b)	100.0	91.7	91.7	80.0
Days until copulation	2.8	2.6	2.8	4.7
a) Copulation index (%)=(Number of copulated animals/number of mated animals) x 100 b) Fertility index (%)=(Number of pregnant animals/number of copulated females) x 100 Values are expressed as mean

 

Delivery and litter data inrats treated orally with Tetrabutylamrnonium bromide the combined repeat dose and reproductive/developmental toxicity screening test
	0 mg/kg	60 mg/kg	180 mg/kg	600 mg/kg
Number of females examined	12	11	11	8
Number of females with live pups	12	11	11	7
Gestation index (%) a)	100.0	100.0	100.0	87.5
Gestation period	22.3	22.2	22.1	21.9
Number of corpora lutea	15.3	15.3	16.3	16.6
Number of implantation sites	14.3	14.3	15.1	15.6
Implantation index (%) b)	93.6	92.9	93.0	93.7
Delivery index (%) c)	100.0	100.0	100.0	87.5
Number of live born pups	13.4	13.5	14.0	14.4
Number of stillborn pups (%) d)	0	0	0	1.9*
Number of live pups on day 0	13.4	13.5	14.0	14.4
Number of pups with external abnormalities (%) e)	0	0	0	0
Live birth index (%) f)	100.0	100.0	100.0	98.2*
Sex ratio of total number of pups at birth g)	81/161 0.50	86/149 0.57	71/154 0.47	52/101 0.53
Sex ratio of live born pups at birth h)	81/161 0.50	86/149 0.57	71/154 0.47	53/103 0.53
Number of live pups on day 4	13.1	13.2	13.7	12.4
Viability index on day 4(%) i)	97.8	97.5	98.1	86.7**
Sex ratio of live pups on day 4 j)	80/157 0.51	84/145 0.57	70/151 0.47	47/87 0.55
Bodyweight of pups (g)
on day 0 male	6.8	6.5	6.7	5.9**
female	6.4	6.0	6.2	5.8**
on day4 male	10.2	10.1	10.0	8.2**
female	9.6	9.2	9.5	7.9**
a)      Gestationindex %)=(Number of females with liveborn/number of pregnant females) X 100 b)      Implantation index(%)=(number of implantations/ number of corpora lutea) X 100 c)      Delivery index(%)=(Number of females which delivered livepups/number of pregnant females) X100 d)      Number of stillborn pups(%)=(Number of stillborn pups/number of stillborn and liveborn pups) X 100 e)      Number of pups with external abnormalities (%)=(Number of liveborn pups with external abnormalities/number of liveborn pups) X 100 f)        Live birth index(%)=(Number of liveborn pups/Total number of pups at birth) X100 g)      Sex ratio of total number of pups on birth=Total number of males/Totalnumber of pups h)      Sex ratio of liveborn pups at birth=Number of liveborn males/Total number of live bornpups i)        Viability index on day 4 after birth (%)=(Number of livepups on day 4 afte rbirth/number of liveborn pups) X100 j)        Sex ratio of livepups on day 4 after birth=Number of live males on day 4 after birth/Total number of live pups on day 4 after birth  Values are expressed as Mean Significant difference from control group; *P<0.05; **P<0.01

Applicant's summary and conclusion

Conclusions:

The study-derived NOAEL for parental general toxicity, reproductive toxicity, and developmental toxicity was 180 mg/kg bw/day. Effects on general parental toxicity reported at 600 mg/kg bw/day included increased mortality rate (8.33% in each sex), transitory changes in body weight (decrease in males, increae in females) and food intake, transitory changes in clinical chemistry, gross findings that showed reversibility, and hypertrophy of perilobular hepatocytes. Effects on reproduction/development reported at 600 mg/kg bw/day included abnormal oestrous cyclicity, increased number of days until copulation, decreased copulation index, decreased insemination index, decreased fertility index, increased stillborn index, decreased pup viability, and decreased pup body weights. Given the nature and number of different effects reported on reproduction and development, which inter alia included significant decreases in live birth index and pup viability, the observed effects on reproduction/development were not considered to be completely secondary to maternal systemic toxicity.

Executive summary:

The test material (with a purity of 100.0% of the substance) was given by oral gavage to 12 Sprague Dawley (Crl:CD) rats per sex per dose at 0, 60, 180 and 600 mg/kg bw/day. Male rats were treated for 2 weeks before mating, for a total of 42 days. Female rats were treated 2 weeks before mating and up to day 4 of lactation, for a total of 41-53 days. Recovery groups of 5 Sprague Dawley (Crl:CD) rats per sex per dose were treated at 0 and 600 mg/kg bw/day for a total of 42 days, and these animals were allowed to recover for 14 days after the final dose. Rats in recovery groups were not used for mating and were included to study reversibility of effects (if any), persistence of effects (if any) and delayed occurrence of effects (if any). Water was used as vehicle. The study was performed according to OECD 422 and GLP. Deaths occurred in 1 male and 1 pregnant female in the 600 mg/kg group. Before death, the male showed fracture of incisors, soft stool and a decrease in the amount of faeces and the female showed staining of lower abdominal fur and staggering gait. In survivors, some males in the 600 mg/kg group showed red urine sporadically and light brown urine was observed at urinalysis, but red urine was a transient change. In the 600 mg/kg group, males showed suppressed body weight gain and females showed tendencies toward high values in body weight and food consumption during the administration period. A high value in food consumption was also observed in females in the 180 mg/kg group. Males in the 600 mg/kg group also showed a low value in body weight during the recovery period. However, since the body weight gain in this group during the recovery period was comparable to that of the control group, they were thought to have recovery. In blood chemistry examination, males in the 600 mg/kg group showed a high value in AST and a low value in blood urea nitrogen and females showed high values in AST and LDH and low values in blood urea nitrogen and creatinine. The females in the 600 mg/kg group showed a high value in calcium as well. These changes were not observed after the end of the recovery period showing reversibility of the changes. Pathological examination showed changes attributable to administration of the test article: in intestinal tracts in males and females in the 180 and 600 mg/kg groups and in the liver in males and females in the 600 mg/kg group. Macroscopically, dilatation in lumina of the cecum was observed. Histologically, diffuse hyperplasia in mucosa in the cecum, which was thought to be related to the macroscopic dilatation in lumina of the cecum, was observed. In the rectum, cell debris in crypt was observed in females. In the recovery group, the above changes were no longer observed and thus recovery from these changes was indicated. In the recovery group, cell infiltration in mucosa was observed in the cecum, colon and rectum. In the liver, hypertrophy of perilobular hepatocytes was observed. There were no test article-related changes in the detailed observation of clinical signs, functional tests, measurement of grip strength or haematological examination. In parent animals in the 60 and 180 mg/kg groups, there were no test article-related changes in the number of days until copulation, copulation index, insemination index or fertility index. There were no test article-related changes in the delivery index, length of gestation period, number of corpora lutea, number of implantation sites, implantation index, number of live born pups or sex ratio, and there were no test article-related changes in parturition condition or lactation observations. In the examination of newborn pups, there were no test article-related changes in external observation or necropsy on day 4 after birth. In the 600 mg/kg group, the number of females that showed abnormalities in oestrous cycle tended to be high, the number of days until copulation tended to be low, copulation index, insemination index or fertility index tended to be low values, a high value in stillborn index, and low values in viability of pups (day 0 and day 4 of lactation) and low values in the body weight of males and females at the time of birth and on day 4 after birth, and male and female pups showed a low value in the body weight gain during the lactation period.

The study-derived NOAEL for parental general toxicity, reproductive toxicity, and developmental toxicity was 180 mg/kg bw/day. Effects on general parental toxicity reported at 600 mg/kg bw/day included increased mortality rate (8.33% in each sex), transitory changes in body weight (decrease in males, increase in females) and food intake, transitory changes in clinical chemistry, gross findings that showed reversibility, and hypertrophy of perilobular hepatocytes. Effects on reproduction/development reported at 600 mg/kg bw/day included abnormal oestrous cyclicity, increased number of days until copulation, decreased copulation index, decreased insemination index, decreased fertility index, increased stillborn index, decreased pup viability, and decreased pup body weights.

The authors of the study did not comment on the relationship between the severity of general maternal toxicity and the severity of reproductive/developmental toxicity. Given the nature and number of different negative effects reported on reproduction and development, which inter alia included significant decreases in live birth index and pup viability, the observed effects on reproduction/development were not considered to be completely secondary to maternal systemic toxicity.