Tetrabutylammonium bromide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 422 study

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, increaes 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 the 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.

Link to relevant study records

Reference

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

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

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

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

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control Group (G1)

Dose / conc.:

60 mg/kg bw/day

Remarks:

Low Dose Group (G2)

Dose / conc.:

180 mg/kg bw/day

Remarks:

Mid Dose Group (G3)

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

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.

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: 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)

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

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

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

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

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.

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

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

Critical effects observed:

no

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

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Study 1:

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, increaes 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 the 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.

Effects on developmental toxicity

Description of key information

OECD 414 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.

Link to relevant study records

Reference

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.

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

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

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

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

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.

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

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.

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

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.

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

Key result

Abnormalities:

no effects observed

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

    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

 

 

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Study 1:

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.

Justification for classification or non-classification

In the OECD 422 study with the substance, statistically/biologically significant effects on reproduction/development reported at 600 mg/kg bw/day (highest dose tested) 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. These effects were observed in the presence of maternal systemic toxicity, which notably included one death (i.e. 8.3% mortality rate). The observed effects on reproduction and development may to some degree have been secondary to maternal systemic toxicity, but most likely not completely. In the OECD 414 study, negative effects that were deemed biologically significant by the test facility were observed at 400 mg/kg bw/day (highest dose tested) and included changes in early resorptions, late resorptions, and post-implantation losses. These effects were observed in the absence of maternal systemic toxicity, but they could not be correlated with any adverse effects on any litter parameter or any other developmental parameter. Considered the results from both the OECD 422 study and the OECD 414 study, the substance is regarded to be classified for Toxicity to Reproduction (Category 2) according to Regulation EC No 1272/2008 by using the following hazard statement: H361fd “Suspected of damaging fertility or the unborn child”.