Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

post-natal screening test

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

March 11th to August 23rd, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is given in section 13 of IUCLID.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Principles of method if other than guideline:

The study screens chemicals for embryonic, fetal, and neonatal toxic responses using pregnant mice treated during major organogenesis (days 6-15 of gestation) by using the postnatal mouse screening test (Chernoff and Kavlock,1982 and 1983)

Chernoff, N. and Kavlock, R. J Toxicol Environ Health 10:541, 1982;
Chernoff, N., Kavlock, R. Short-Term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum Publishing Co., NY, 1983 pp 417-427.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-Nitrilotriethanol

Test animals

Species:

mouse

Strain:

CD-1

Remarks:

albino

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, New York).
- Females nulliparous and non-pregnant: virgin female (Phase I) and primigravida (Phase II and III).
- Age at study arrival: 6-8 weeks
- Weight at treatment initiation: 23.8-25.5 g (Phase I), 26.55 - 28.32 g (Phase II, only pregnant mice), 27.1-27.9 (Phase III, only pregnant mice).
- Housing: individually housed in polycarbonate shoe box cages with stainless steel tops and hardwood bedding (Ab-Sorb-Dri, Lab Products, Maywood, N.J.). Cages were sanitized and fresh bedding was supplied at least once during the study.
- Diet: Purina Certified Rodent Chow #5002, ad libitum. Ziegler Brothers NIH-07 Rodent Chow (Gardners, PA), was supplied to the Phase III animals on the day they arrived at the test facility. Purina Certified Rodent Chow H5002 was made available to the mice later the same evening.
- Water: fresh water, ad libitum.
- Quarantine period: the mice were quarantined for five days in the room in which the study was to be conducted. This shortened quarantine period was used in Phase I to parallel that used in Phases II and III with pregnant mice. During this period, observations were performed twice daily for mortality and general physical appearance.

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F, 23 ± 3 °C.
- Photoperiod: 12-hour light/dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: appropriate amounts of the compound were weighed on a pan balance (accurate to 0.1 mg). Distilled water was then added and the resulting test solution mixed on a magnetic stirrer until suspended. The daily dosing solutions were prepared and were stored refrigerated in amber glass vials to prevent photodegradation. On the day of dosing, an aliquot was allowed to warm to room temperature prior to dosing. All dosing concentrations were verified as accurate by the chemistry staff. The dosing mixtures were thoroughly vortexed just prior to and intermittently during dosing.

ADMINISTRATION OF THE TEST SOLUTION: the test dosing solution was delivered directly into the stomach via a 20 gauge 1 1/2 inch stainless steel gavage needle attached to a 1 cc syringe.

VEHICLE
- Amount of vehicle: 10 ml/kg bw

Details on mating procedure:

PHASE III
Beginning on day 18 of gestation and continuing until all litters were delivered, females were observed twice daily for evidence of labor and delivery of litters. Observations were made before 9:00 a.m. and after 4:00 p.m. The day of delivery was recorded to the nearest half day. The time, date, and gestation day were recorded when labor, fetuses, or other evidence of delivery was observed. Females were not disturbed if they were in labor or if they had not finished cleaning newborn pups. The delivery of pups was considered complete if the pups were clean and dry.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to and upon completion of the dosing interval, the dosing solutions were analyzed to document concentration and chemical stability. The substance was analyzed by gas chromatography in the following system conditions:
Detector: electron capture.
Detector Temperature: 250 °C.
Injection Temperature: 250 °C.
Flow rate: 30 ml/min, 95 % Argon, 5 % Methane.
Concentration range: 1 mg/ml in methanol.

Duration of treatment / exposure:

five days (Phase I), ten days:gestation days 6-15 (Phase II and III).

Frequency of treatment:

once daily, over a five-day period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 125 mg/kg bw/day

Remarks:

Phase III - Reproductive test

Doses / concentrations 2

Dose / conc.:

9 600 mg/kg bw/day

Remarks:

Phase II - Range finding

Doses / concentrations 3

Dose / conc.:

4 800 mg/kg bw/day

Remarks:

Phase II - Range finding

Doses / concentrations 4

Dose / conc.:

2 400 mg/kg bw/day

Remarks:

Phase II - Range finding

Doses / concentrations 5

Dose / conc.:

1 200 mg/kg bw/day

Remarks:

Phase II - Range finding

Doses / concentrations 6

Dose / conc.:

600 mg/kg bw/day

Remarks:

Phase II - Range finding

Doses / concentrations 7

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Phase I - Range finding

Doses / concentrations 8

Dose / conc.:

100 mg/kg bw/day

Remarks:

Phase I - Range finding

Doses / concentrations 9

Dose / conc.:

10 mg/kg bw/day

Remarks:

Phase I - Range finding

No. of animals per sex per dose:

3 virgin females (Phase I), 4 mated females (Phase II), and 50 mated females (Phase III).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the substance did not produce chemical related mortality when administered at dose levels of 10, 100, and 1000 mg/kg/day in Phase I. Therefore, the dose levels for the Phase II selected were 600, 1200, 2400, 4800, and 9600 mg/kg/day. Based on the probit analysis of combined mortality data from the Phase I and II tests, the recommended doses for the Phase III reproductive study (LD10) was 1125 mg/kg/day.

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes.
- Time schedule: twice daily; once in the morning and once in the afternoon. Observation for signs of toxicity and mortality were also performed once on the days the animals were not dosed (7 days post treatment for Phase I, 2 days post treatment for Phase II).
- Parameters checked: signs of toxicity, mortality. All animals which succumbed during the dosing phase were examined for evidence of dosing error. Dosing error deaths were determined when: compound was found in the thoracic cavity; compound was found in the lungs and/or a hole was found in the esophagus. All other deaths were assumed to be treatment related.

BODY WEIGHT: Yes.
- Time schedule for examinations: body weights were measured within the next two days after receipt of the animals. Phase I animals were recorded at the time of randomization, on days 1 and 5 of treatment and on days 3 and 7 after the final dose. Phase II and III animal body weights were recorded at the time of randomization, on days 6 through 15 of gestation and on day 17 of gestation. Additional body weights measured for Phase III were recorded on post partum day 0 and post partum day 3. After delivery was complete, the dam was removed and weighed then placed temporarily in a holding container. Body weight changes were calculated for each phase of the study.

Litter observations:

PARAMETERS EXAMINED: the number of live and dead pups, birth weights (of all live pups), and growth and survival to 3.5 days of age are the primary response variables measured. The live pups of a litter were weighed together to give a litter weight, and the average litter weight was calculated.

GROSS EXAMINATION OF DEAD PUPS: dead fetuses were discarded.

Postmortem examinations (parental animals):

SACRIFICE: all surviving mice (at post partum Day 3) were sacrificed by carbon dioxide asphyxiation following the collection of terminal body weights. On gestation day 17, all surviving Phase II mice were sacrificed. Any female from Phase III that did not show signs of delivering a litter by gestation day 22 was sacrificed.

EXAMINATIONS:
Phase I: the uteri were examined for all Phase II mice. Each female was classified as pregnant or non pregnant. Pregnant mice were noted as having live fetuses or no live fetuses.
Phase III: the uteris were examined for evidence of pregnancy for Phase III mice that had not delivered by gestation day 22. If there was no gross evidence of pregnancy, the uterus was placed in a 10 % ammonium sulfide solution to make early implantation sites visible. Based on the presence of early implantation sites, the female was classified as having been either pregnant or never pregnant.

Postmortem examinations (offspring):

SACRIFICE: at postpartum Day 3 the litters were sacrificed and discarded.

Statistics:

An overall test for homogeneity of variance (Bartlett's test) and F-test were performed on the weight data of each group following randomization.
Average body weight per group and average body weight change per group were calculated for treatment and control groups. Probit analysis of mortality and morbidity data generated in Phase II of the range finding study was used to determine the predicted LD10 for the Phase III.
ANOVA was used for anaylizing random weights, Fisher's exact test (one tail) for survival (each group vs control, all dams and pregnant only) and for proportion of viable litters, Chi-Square test (one-tail) for analyzing data of pups survival. Mann-Whitney U-Test (2-tail) was used for analyzing data of weight gains and to compare each group to the concurrent control for number of live pups/litter, length of gestation, average pup weight (day 0, day 3) and average weight gain/litter (day 3-day 0). The p-value was not corrected for multiple comparisons.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Phase III: one animal was noted as having a subcutaneous lump (treatment day 2). This did not appear to be related to the treatment. One other animal exhibited signs of toxicity throughout the treatment and post-treatment period. These signs included: hunched posture, languid behavior, rough haircoat, wheezing, labored respiration, head tilt, squinted eyes, and pale extremities. All other animals appeared normal throughout the treatment and post-treatment period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Phase III: no deaths occured

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Phase III: the animals were significantly heavier from the control group at postpartum day 0 (p=0.02) but not at postpartum day 3 (p=0.43). The average weight gain from pretreatment to post partum day 0 was 5.3 grams. The dams were significantly greater.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No statistically significant group differences were noted in the length of gestation; no significant differences on the reproductive index compared to control group.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks on result:

other: not determined/specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No statistically significant effect of the substance was observed upon the percent viable litters and upon pup survival.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in the average weight of each live pup/litter.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

other: not determined/specified

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

The number of animals found dead or sacrificed as well as the number of pregnant animals and live pups in the control and tested group in Phase III, is presented in the table below.

Table: maternal status of animals in Phase III.

Maternal status
	Water	Test material
Number evaluated	49	50
Animals found dead	0	0
Pregnant	0	0
Never Pregnant^a	0	0
Animals sacrificed ^b	49	50
Delivered Live pups	25	31
No live pups ^c	3	3
Never pregnant ^a	21	16

a = Ammonium Sulfide Negative

b = Number of animals sacrificed - includes animals sacrificed day 3 post partum and animals sacrificed on gestation day 22

c = Visually dead pups or ammonium sulfide positive

The average maternal body weight changes in the control and treatment group for Phase III, are presented in the table below.

Table: average maternal body weight, in grams (mean ± s.d)

	Vehicle	Test material
Random weight
Pregnant only	26.1 ± 1.15^a	26.0 ± 1.11
n^b	28	28
Random weight
Viable litters only	26.0 ± 1.13	26.1 ± 1.09
n	25	31
Weight gain
Postpartum- randomization	+ 4.3 ± 1.60	+ 5.3 ± 1.59*
n	25	31

a = an analysis of variance on the results was performed, * denoting p=0.02 and ** denoting p<0.01.

b = n = number of animals

The Reproductive and Litter data for the control and treated group for Phase III, are presented in the table below.

Table: reproductive and litter data

	Reproductive Ratio	Index^a Percent	Average number/litter		Postnatal Ratio	Survival^b Percent
			Day 0 Live	Day 3 Live
Vehicle	25/28	89.3^c	10.2 ± 3.1	10.1 ± 3.1	242/255	94.9
n^d			25	25
Test material 1125 mg/kg	31/34	91.2	9.6 ± 2.8	9.5 ± 2.8	278/298	93.3
n			31	31

a = Number of females producing viable litters/number of surviving females that were ever pregnant

b = Number of pups alive on day 3/number of pups alive

c = Reproductive index and post natal survival results test with *** denoting (p ≤0.001). The number of live pups per litter were analyzed by the Mann-Whitney U-test with *** denoting (p ≤0.001). None of the other results had a value of less than 0.05.

d= Number of litters used to calculate the mean

The duration of gestation (in days) of dams in Phase III is presented in the table below for both control and treated group.

Table: duration of gestation (in days)

	Vehicle	Test material
Mean ± Standard deviation	18.2^a ± 0.35	18.3 ± 0.34
n^b	25	31

a = Comparison of exposed groups to vehicle control group was evaluated by Mann-Whitney U-test with *** denoting (p ≤0.001).

c = number of animals that produced viable litters

The average weight of each live pup per litter and Day 3 average live pup weights minus day 0 average live pup weights (g) for control and treated group are presented in the table below.

Table: average weight of pup and litter and weight change for animals in control and treated groups in Phase III.

	Average weight/pup/litter (g)		Change
	Day 0 Live	Day 3 Live	Day 3 - Day 0
Vehicle	1.5 ± 0.19^a	2.1 ± 0.32	0.6 ± 0.18
n^b	25	25	25
Test material 1125 mg/kg	1.6 ± 0.20	2.2 ± 0.30	0.6 ± 0.23
n	31	31	31

a = Results were analyzed by Mann-Whitney U-test with ***denoting (p ≤0.001). There were no other statistically significant differences among the groups, (p ≤0.05).

b = Number of litters with live pups

The results of the analysis of dosing solutions used in Phase III, before and after dosing were as follows:

Before dosing: 113.27 mg/ml

After dosing: 109.43 mg/ml

Applicant's summary and conclusion

Conclusions:

The substance did not produce any adverse reproductive effects.

Executive summary:

The potential of the substance to cause adverse reproductive effects was evaluated by the Chernoff/Kavlock post-natal mouse screening test. The screen consisted of three experimental phases. Phases I and II are range-finding studies designed as a method to identify the predicted LD10, to be used in the Phase III. In phase I three non-pregnant mice were exposed to 3 concentrations of the substance for 5 concecutive days, while in phase II the substance was administered to 4 mated females on gestation days 6 -15 in 5 dose levels. In phase III (reproductive test) 50 mated females were exposed to 1125 mg/kg/day of the substance on gestation days 6 -15. Reproductive hazard potential was evaluated by consideration of the maternal body weight, maternal mortality, signs of toxicity, pup counts at birth (live and dead), pup weights and offspring survival from birth to day 3 postpartum.

The substance did not affect maternal mortality, the number of viable litters, litter size, percent survival of pups, birth weight of the pups and weight gain by the pups; it did not produce any evidence of developmental toxicity. It is, though, recommended that the substance be retested in the Chernoff/Kavlock test at a higher dose which results in approximately 10 % maternal mortality. Until it is retested, triethanolamine is judged to be inaccurately tested in this test.