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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
October 20 - November 20, 1997 to January 30, 1998
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from NTRL report

Data source

Reference
Reference Type:
other: NTRL
Title:
SUPPORT: FINAL REPORT, DEVELOPMENTAL TOXICITY EVALUATION OF TERGITOL NP-4 SURFACTANT ADMINISTERED BY GAVAGE TO CD(SPRAGUE-DAWLEY) RATS, WITH COVER LETTER DATED 4/23/1999
Author:
Rochelle W. Tyl, Ph.D., DABT Melissa C. Marr, B.A., LATG Christina B. Myers, M.S.
Year:
1999
Bibliographic source:
UNION CARBIDE CORP, NTRL: OTS0559310-1, 1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Repeated dose oral toxicity of the substance TERGITOL NP-4 was assesed in CD Sprague–Dawley rats.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 4-Nonylphenol, branched, ethoxylated
- Molecular formula : C17H28O2
- Molecular weight : 264.409 g/mol
- Substance type:Organic
- Physical state: Liquid
Specific details on test material used for the study:
- Name of test material : TERGITOL NP-4
- EC name: 4-Nonylphenol, branched, ethoxylated
- Molecular formula : C23H40O5 (SAX)
- Molecular weight : 396.63 g/mol (SAX)
- Substance type: Organic
- Physical state: Transparent, colourless Liquid
- Purity: > 97%
- Impurities (identity and concentrations): polyethylene glycol, <3%, dinonylphenol ethoxylate, <3%,

Test animals

Species:
rat
Strain:
other: CD (Sprague Dawley)
Details on species / strain selection:
No data
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation:
Female rats= 56 days
Male rats= 70 days
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Males were housed singly in solid-bottom polycarbonate cages (8" x 19" x 10.5") with stainless steel wire lids. Non mated females were group housed (maximum three per cage), and mated females were singly housed in solid-bottom polycarbonate cages (8" x 19" x 10.5") with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ). Sani-Chips® animal bedding (P. J. Murphy Forest Products, Montville, NJ) was used in all cages.
- Diet (e.g. ad libitum): Pelleted feed (No. 5002 Purina Certified Rodent Diet®; PMI Feeds, Inc., St. Louis, MO). ad libitum
- Water (e.g. ad libitum): tap water (Durham, NC water system, in plastic bottles with stainless steel sipper tubes) available ad libitum
- Acclimation period: 7 days quarantine periods for the females

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
target temp: 64-79°F (17.8 - 26.1°C)
maintained temp: 70.7-73.2°F (21.5 - 22.9 °C)

- Humidity (%):
target humidity: 30-70%
maintained humidity 35.7 - 63.8%

- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12:12 hour light: dark cycle

IN-LIFE DATES:
From: Dosing: November 3-15,1997 (gestation day 6 through 15)
To: Necropsy: November 17-20,1997 (gestation day 20)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The Tergitol NP-4 Surfactant for each dose was weighed out into a calibrated beaker (400 ml beaker to hold 200 ml, with adjustments for different volumes if needed). The appropriate volume of corn oil was added to each beaker, stirred with a stirring bar until the chemical was no longer visible, and then stirred for ten minutes more to give a dose range of 0, 50, 250 or 500 mg/Kg/day. The resulting formulation was sampled for archive and analytical samples and stored at 1-8°C in sealed, amber glass bottles protected from light. After storage and prior to use, the solutions were allowed to warm to room temperature and then stirred for ten minutes with a stirring bar. The formulations were then used for dosing.

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 50, 250 or 500 mg/Kg/day
- Amount of vehicle (if gavage): 1.0 ml/kg
- Lot/batch no. (if required): 325A7
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to study dose formulation, formulations encompassing the range of dose employed (50 and 600 mg/mL) were assayed for homogeneity, stability, and dose level verification
Duration of treatment / exposure:
10 consecutive days (Gestation day 6 through gestation day 15)
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
0, 50, 250, or 500 mg/kg/day
No. of animals per sex per dose:
Total: 100
0 mg/kg/day=25 sperm positive females
50 mg/kg/day=25 sperm positive females
250 mg/kg/day=25 sperm positive females
500 mg/kg/day=25 sperm positive females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses was based on a range-finding study in pregnant rats performed at RTI. In range finding study, four group of eight sperm positive females were dosed once daily on gestation day 6 through 15 at concentartion 0, 500, 1500, and 3000 mg/kg/day at a dosing volume of 5.0 ml/kg. At 1500 and 3000 mg/kg/day, at least 50% of the dams died. At 500 mg/kg/day, all dams survived and were pregnant; seven of eight dams were pregnant at 0 mg/kg/day. Therefore, the highest dose level for the present study, 500 mg/kg/day, was chosen to induce overt maternal and developmental toxicity.

- Rationale for animal assignment (if not random): Sperm-positive female rats (dams) were assigned to treatment groups by a stratified randomization method, designed to provide uniform mean body weights across dose groups on gd 0 at the initiation ofthe study.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: daily, except during the dosing period when they were made at least twice daily. At least once daily on gd 0-5 (prior to dosing period), on gd 16-20 (postdosing period) at least twice daily, at dosing, and at least once after dosing throughout the dosing period (gd 6 through gd 15).

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were taken on gestational day 0, 6,9,12,15,18, and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, measured for the intervals gestational day 0-6, 6-9, 9-12, 12-15, 15-18, 18-20, 6-15 (treatment period), 15-20 (posttreatment period), and 0-20 (gestational period)..
- 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

FOOD EFFICIENCY: No data
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

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

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, On gd 20, maternal animals were sacrificed by asphyxiation with C02, thoracic and abdominal cavities and organs examined, and their pregnancy status was confirmed by uterine examination. At sacrifice, the body, liver, and uterus of each sperm-positive female were weighed, ovarian corpora lutea counted, and uterine contents (i.e., number of implantation sites, resorptions, dead fetuses, live fetuses) recorded.

HISTOPATHOLOGY: No data available
Other examinations:
Uteri which presented no visible implantation sites were stained with ammonium sulfide (10%) in order to visualize any implantation sites, which may have undergone very early resorption. All live fetuses were weighed, sexed, and examined for external morphological abnormalities, inclUding cleft palate. Approximately one-half of the live fetuses per litter were examined for visceral abnormalities and had their sex confirmed using a fresh tissue dissection method. These fetuses were decapitated prior to dissection, and the heads were fixed in Bouin's solution for free-hand sectioning and examination. All fetal carcasses were eviscerated, macerated, and stained with alcian blue/alizarin red S, and half (intact fetuses) per litter were examined for skeletal malformations and variations. After examination, all maternal and fetal organs and maternal carcasses were destroyed by incineration. Fetal carcasses were stored in glycerin:70% ethanol (1:1) following examination of skeletal structures or for those not examined skeletally; fetal heads were stored in 70% ethanol solution.
Statistics:
As mentioned below

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Details on results:
Clinical signs and mortality:
Mortality: One female at 500 mg/kg/day died on gd 6 after dosing sue to misdirected dose

Clinical signs: Treatment-related clinical observations were present almost exclusively at 250 and 500 mg/kg/day. Rust colored fur on the head, face, and back, a nonspecific indicator of stress (from hemoglobin breakdown products in the Harderian gland excreted via the tear ducts, so-called "bloody tears" or chromodacryorrhea, groomed from the eyes and nose), was observed only at 500 mg/kg/day and only on gd 13 and 15. Piloerection, another nonspecific sign of stress, was observed at 500 mg/kg/day on gd 6 through 20, at 250 mg/kg/day on gd 7-10 and 13-15, and at 50 mglkg/day on gd 14 in one dam, in dose-related patterns of incidence and time to presence. Rough coat (more severe than piloerection, indicative of more stress) was observed only at 500 mg/kg/day on gd 12-15. Rooting in bedding postdosing (most likely due to taste aversion and not toxicity, per se) was observed at all doses on gd 7-15 in a dose-related pattern of incidence. Lethargy bwas observed at 500 mg/kg/day on gd 6-15 and at 250 mg/kg/dayon gd 7 (one dam) and 8 (one dam).

Prone positioning was observed only at 500 mg/kg/day on gd 6-14. Pica was observed at 250 mg/kg/day on gd 6 and 8 and at 500 mg/kg/day on gd 6 and 7. Other clinical observations, such as alopecia (various areas), were unrelated to treatment.

Body weight and weight gain: Maternal body weights were equivalent across all groups for gd 0 and 6 prior to the onset of dosing. Starting on gd 9, mean body weights at 250 and 500 mg/kg/day were statistically significantly reduced for gd 9, 12, 15, and 18. Maternal body weights on gd 20 (in-life and at sacrifice) were unaffected. Maternal weight change was equivalent across all groups for gd 0-6, prior to the onset of dosing. Maternal weight change was significantly reduced at all doses for gd 6-9, the first interval of the dosing period, and was significantly reduced at 500 mg/kg/day for gd 9-12. Maternal weight change for the treatment period (gd 6-15) was significantly reduced at 250 and 500 mg/kg/day. Maternal weight changes were significantly increased at 250 and 500 mg/kg/day for gd 15-18 and 18-20 during the postdosing period. Weight change during the entire postdosing period (gd 15-20) was also significantly increased at 250 and 500 mg/kg/day. Maternal gestational weight change and gestational weight change corrected for the weight of the gravid uterus were unaffected.

Food consumption and compound intake: Feed consumption was statistically significantly reduced at 250 and 500 mg/kg/day for gestation day 6-15 (treatment period), and 0-20 (gestational period). And was significantly increased for gestation day 18-20 at all doses. Maternal feed consumption as g/kgfday was significantly increased at 500 mg/kg/day for gd 18-20 and 15-20 (postdosing period), at 250 mg/kg/day for gd 18-20 and 15-20, and at 50 mg/kgfday for gd 18-20 and 15-20

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No data available

Clinical chemistry: No data available

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: Absolute liver weight and maternal liver weight, relative to terminal body weight, were significantly increased at all doses. However, this finding may be due to induction of hepatic metabolizing enzymes and concomitant increase in liver mass and not to toxicityGravid uterine weight was unaffected.

Gross pathology: Necropsy findings for unscheduled deaths (one at 500 mg/kg/day) included blood present in urogenital opening, urinary bladder, mouth and nose, and lung lobes hemorrhagic and edematous. At scheduled necropsy on gd 20, one dam at 50 mg/kg/day exhibited an area of extra hard tissue on left median liver lobe.

Histopathology: No data available

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No significant alterations were observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Summary and Statistical Analysis of Matemal Body Weights, Weight Changes, Organ Weights and Relative Organ Weights

 

Tergitol (mg/kg/day, po)

 

0

50

250

500

No. of animals

25

25

25

25

No. of Removed

0

1a

0

0

No. found dead

0

0

0

1b

Maternal body weight (gd 0) (g)

227.6 ± 2.3

226.9±2.2

227.0± 2.3

228.7±2.4

Maternal body weight (gd 20 at sacrifice) (g)

365.4±3.3

370.2±4.4

365.5±3.1

358.1±4.2

Gravid Uterine weight (g)

82.54±1.86

82.30±2.44

85.54±2.40

82.99±2.96

Maternal Liver weight (g)

15.93±0.21

16.69±0.25**

16.74±0.21*

17.16±0.25**

Relative Maternal Liver weight (g)

4.36±0.04

4.58±0.04

4.58±0.04

4.80±0.07

Maternal feed consumtion (gd 0-6) (g/day)

22.2±05

23.0±0.5

22.3±0.5

22.9±0.5

Maternal feed consumtion (gd 0-20) (g/kg/day)

77.8±0.7

80.1±0.7

75.4±1.0

72.7±0.9**

aDam 98 was removed because she delivered on gestational day 19

bDam 63 was found dead on gestational day 6 after dosing

*p<0.05 Dunnett's Test

**p<0.01 Dunnett's Test

 

 

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for TERGITOL NP-4 is considered to be 50 mg/kg/day in CD Sprague–Dawley rats.
Executive summary:

Repeated dose oral toxicity study for substanceTERGITOL NP-4 (EC name: 4-Nonylphenol, branched, ethoxylated) was conducted intimed-pregnant CD (Sprague-Dawley) rats. Based on the range finding study dose were selected. Tergitol NP-4 Surfactant, dissolved in corn oil and administered by gavage once daily on gestational days (gd) 6 through 15, to 4 group of 25 sperm-positive at doses of 0,50, 250, or 500 mg/kg/day at a dosing volume of 1.0 ml/kg. The rats were observed for mortality, clincal signs, body weight and feed consumption changes, gross pathology and organ weight changes. One pregnant female rat died at 500 mg/kg/day. Maternal body weights were significantly reduced at all doses and body weight gain were significantly reduced at 250 and 500 mg/kg/day and No effect observed on gestational weight gain, corrected for gravid uterine weight, and gravid uterine weight. Increased absolute and relative liver weight was observed. However, this finding may be due to induction of hepatic metabolizing enzymes and concomitant increase in liver mass and not to toxicity. Maternal treatment-related clinical signs were observed at 250 and 500 mg/kg/day. Maternal feed consumption was significantly reduced at 250 and 500 mg/kg/day and was significantly increased at all doses for gd 18-20. Fetal related observations like increased incidences of fetal skeletal variations, specifically bilateral and unilateral rudimentary ribs on Lumbar I increased at 250 and 500 mgl1<g/day, and normal cartilage, bipartite ossification center in a thoracic centrum apparently increased at 500 mg/kg/day, with no statistically significant difference. The incidence of fetal cleft palate was also observed but was not statistically significantly different among groups. These findings were observed in the absence of any treatment-related effects on pre or postimplantation loss, live litter size, sex ratio (% males), or on fetal body weight per litter. The No Observed Adverse Effect Level (NOAEL) for TERGITOL NP-4 is considered to be 50 mg/kg/day in CD Sprague–Dawley rats.