Lead 2,4,6-trinitro-m-phenylene dioxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

85 days

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented and corresponded to the criteria set for assessing animal studies in (Klimisch, et. al. 1996)- A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data, giving due consideration to the published data quality criteria in place at the time the study was conducted.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Principles of method if other than guideline:

Since the populations living in lead -contaminated environments are likely to be exposed to the metal continuously from conception until death, the authors have developed a rat "lifetime" lead exposure model that reproduces the reproductive toxicity and growth effects observed clinically in lead poisoning (Ronis, 1996).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, lead salt, basic

EC Number:

257-175-3

EC Name:

Acetic acid, lead salt, basic

Cas Number:

51404-69-4

Details on test material:

Lead acetate (ACS plus grade) were obtained 99.8% pure) was purchased from Sigma Chemical Co. (St. Louis, MO).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan industries (Indianapolis, IN)
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: They were housed in an AAALAC-approved facility
- Diet (e.g. ad libitum): Rats were given ad libitum access to soy-grain-based laboratory feed (Formulab diet 5008, PMI Feeds, Inc., St. Louis, MO).
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Constant Temperature
- Humidity (%): Constant Humidity
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Lights on between 6:00 AM and 18:00 PM

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Time-impregnated female Sprague-Dawley rats (n=10-15/group) were exposed to either lead acetate in the drinking water at levels of .05%, 0.15%, or 0.45% or an acetic acid solution containing an equivalent amount of acetate to the highest lead dose (0%) (n = 10-15/lead treatment group). One milliter of 5N HCL was added to each leter of each solution to preclude precipitation of insoluble lead salts.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

It was previously demonstrated that developmental exposure of rats to as much as 0.6% (w/v) lead acetate produces reproductive toxicty with no apparent compromises to the health of the animals and that exposure to acetic acid at this dose has no effect on any reproductive parameter (Ronis et al. 1996, 1997).

Details on mating procedure:

Time-impregnated Sprague Dawley Rats

Duration of treatment / exposure:

Dams had access to water containing lead acetate or acetic acid until weaning, and thereafter pups received lead acetate or acetic acid until euthanization.

Duration of test:

One male and one female pup per litter were euthanized at age 21 days (weaning), 35 days (early puberty), 55 days (late puberty), and 85 days (adulthood).

Doses / concentrations

Remarks:

Doses / Concentrations:
Time-impregnated female Sprague-Dawley rats (n=10-15/group) were exposed to either lead acetate in the drinking water at levels of .05%, 0.15%, or 0.45%
Basis:
nominal in water

No. of animals per sex per dose:

37 rats, 21 days, 35 days, 55 days, 85 days. Four male and four female pups per litter.

Control animals:

yes

Details on study design:

A dose-response study was conducted in a rat model to examine the effects of lifetime lead exposure on the development of the reproductive system and the endocrine mechanisms underlying these effects. Time-impregnated female Sprague-Dawley rats (n = 10-15/group) were exposed to lead acetate in the drinking water at levels of 0.05%, 0.15%, or 0.45% (w/v) initiated on gestational day 5. At birth, litters were culled to four male and four female pups. Exposure of dams to lead was continued until weaning, following which, the pups continued to be exposed to lead acetate in drinking water until sacrifice. One male and one female pup from each letter were sacrificed at age 21, 35, 55, and 85 days.

Examinations

Maternal examinations:

Lead concentrations in whole blood and plasma were measured in dams sacrificed at the end of lactation.

Fetal examinations:

At birth litters were sexed, weighed, and crown-to-rump length (CRL) and anogenital distance (AGD) were measured. Excess pups were removed from litters at random to cull the numbers down to four male and four female pups in each litter. Trunk blood was collected and pooled from culled male and female pups in each litter for determination of whole-blood lead and plasma sex sterouids. Dams had access to water containing lead acetate or acetic acid until euthanization. Pups were weighed every 5 days, and food and water intake were recorded. Vaginal opening was monitored in female pups from age 30 days, and estrus cycling was determined by histological examination of vaginal smears form age 60 days. One male and one female pup per litter were euthanized at age 21 days (weaning), 35 days (early puberty), 55 days (late puberty), and 85 days (adulthood).

Statistics:

One pup of each sex was sacrificed from each litter at each time point to take litter effects into account. For birth weight, crown-to-rump length. anogenital distance, pup motality, relative prostate weight, and plasma and pituitary hormone concentrations, statistical significance, was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis with p<.06 considered significant. For vaginal opening data, statistical significance was conducted using the Wilcoxson rank sum test with p<.05 considered significant. Since not all of the dams on test were pregnant, data are presented as mean +/- SEM for pups derived from n=5 (0%), n=10 (0.05%), n=8 (0.15%), and n=9 (o.45% lead acetate) litters except for plasma sex steroid detrmination at birth and data derived from males at age 85 days. Neonatal testosterone values were determined on n= 5-6 pools of plasma each derived from 3-5 pups from 1-2 litters. Neonatal plasma estradiol values were determined from pooled samples each derived from all the excess female pups culled from each treatment group (approximately 20 animals). Data for male pups at age 85 days is presented as mean +/- SEM for pups derived from n=3 (0% control) litters.

Indices:

Blood lead levels, reproductive physiology, hormone analysis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:not examined

Effect levels (maternal animals)

Dose descriptor:

LOEL

Effect level:

ca. 0.05 other: % (w/v)

Based on:

dissolved

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

A significant dose response decrease in birth weight and crown-to-rump length was observed in all lead-exposed litter. However, no marked effects were observed on anogenital distance/crown-to-rump length ratios. Lead exposure resulted in a delay in sexual maturity as measured by prostate weight in male pups and time of vaginal opening in femle pups, which increased with lead dose. These disruptions in reproductive physiology were accompanied by a significant decrease in neonatal sex steroid levels and suppression of the plasma concentrations of testosterone (male) and estradiol (female) during puberty. In male pups this was accompanied by a significant decrease in plasm luteinizing hormone (LH), elevated pituitary LH content, and a decrease in testosterone/LH ratios at the highest dose. In female pups, although no effects were observed on plasma LH concentration, a similar significant elevation in pituitary LH content was observed during early puberty. Postpuberty, plasma LH and sex steroid concentrations were unaffected at any dose in spite of continued lead exposure. No significant effects were observed on epididymal sperm count in male pups at 85 days of age. In female pups, estrus cycling was only significantly disrupted at the highest dose of lead. These data suggest that the reproductive axis is particularly sensitive to lead during specific developmental periods, resulting in delayed sexual maturation produced by suppression by sex steroid biosynthesis.

Birth data for pups exposed to lead acetate in utero

Treatment*	Gestation**	Pups***	M/F****	Dead*****	Body weight******	GRL*******	AGD********	AGD/CRL*********
0%	21.2 +/- 0.2	11 +/-2	1.2	4 +/- 3	6.6 +/- 0.2(M); 6.3 +/- 0.2 (F)	40.9 +/- 0.9(M); 39.4 +/- 0.5 (F)	3.08 +/- 0.1 (M); 1.74 +/- 0.1 (F)	7.53 +/- 0.3 (M); 4.42 +/- 0.1
0.05%	21.8 +/- 0.2	9 +/- 1	0.8	3 +/- 3	6.5 +/- 0.2 (M); 6.4 +/- 0.3 (F)	40.7 +/- (M); 39.9 +/- 0.3 (F)	2.95 +/- 0.2 (M);1.56 +/- 0.1	7.25 +/- 0.4 (M); 3.91 +/- 0.2 (F)
0.15%	21.8 +/- 0.2	9 +/- 1	0.8	10 +/- 6	6.0 +/- 0.2*********8(M);5.9 +/- 0.3 (F)	38.3 +/- 0.8**********(M);38.2 +/- 0.7(F)	2.60 +/- 0.1(M)**********;1.47 +/-0.1**********	6.79 +/- 0.1***********(M);3.85 +/_ 0.1**********(F)
0.45%	21.8 +/- 0.3	8 +/-1	1.0	28 +/-8**********	5.2 +/- 0.3(M)**********;4.7 +/- 0.2**********(F)	36.5 +/- 0.8**********(M); 35.4 +/- 0.7**********(F)	2.61 +/- 0.2**********(M);1.43 +/- 0.7**********(F)	7.15 +/- 0.4(M)/4.03 +/- 0.07(F)
Note: data presented as mean +/- SEM for pups derived from n= 5 (0%), n= 10 (0.05%), n=8 (0.15%), and n = 9 (0.45% lead acetate) litters. Statistical comparison by one-way ANOVA followed by Dunnett's posthoc analysis.	*Lead acetate exposure (% w/v) of dams in drinking water ad libitum from gestational day 5 until parturition.** length of gestation (days).***Number of pups/litter prior to culling.**** Ratio of male to female pups/litter.*****Pup mortality (%).	******Birth weight (g/pup); (M) male pups, (F) female pups.*******Crown-to-rump length (MM)********Anogenital distance (MM).*********Anogenital distance/crown-to-rump length ratio**********Significant at p < .05 vs. 0% control

Applicant's summary and conclusion

Conclusions:

These data suggest that the reproductive axis is particularly sensitive to lead during specific developmental periods, resulting in delayed sexual maturation produced by suppression by sex steroid biosynthesis. The mechanisms underlying this appear to involve lead actions on both LH release and gonadal function. At low, environmentally relevant blood lead concentrations, adaptation to the continuous presence of the metal ion occurs and surprisingly little effect is observed on adult reproductive endocrinology and physiology.

Executive summary:

A dose-response study was conducted in a rat model to examine the effects of lifetime lead exposure on the development of the reproductive system and the endocrine mechanisms underlying these effects. Time-impregnated female Sprague-Dawley rats (n = 10-15/group) were exposed to lead acetate in the drinking water at levels of 0.05%, 0.15%, or 0.45% (w/v) initiated on gestational day 5. At birth, litters were culled to four male and four female pups. Exposure of dams to lead was continued until weaning, following which, the pups continued to be exposed to lead acetate in drinking water until sacrifice. One male and one female pup from each letter were sacrificed at age 21, 35, 55, and 85 days. A dose-response study was conducted in a rat model to examine the effects of lifetime lead exposure on the development of the reproductive system and the endocrine mechanisms underlying these effects. Time-impregnated female Sprague-Dawley rats (n = 10-15/group) were exposed to lead acetate in the drinking water at levels of 0.05%, 0.15%, or 0.45% (w/v) initiated on gestational day 5. At birth, litters were culled to four male and four female pups. Exposure of dams to lead was continued until weaning, following which, the pups continued to be exposed to lead acetate in drinking water until sacrifice. One male and one female pup from each letter were sacrificed at age 21, 35, 55, and 85 days.

A significant dose response decrease in birth weight and crown-to-rump length was observed in all lead-exposed litter. However, no marked effects were observed on anogenital distance/crown-to-rump length ratios. Lead exposure resulted in a delay in sexual maturity as measured by prostate weight in male pups and time of vaginal opening in femle pups, which increased with lead dose. These disruptions in reproductive physiology were accompanied by a significant decrease in neonatal sex steroid levels and suppression of the plasma concentrations of testosterone (male) and estradiol (female) during puberty. In male pups this was accompanied by a significant decrease in plasm luteinizing hormone (LH), elevated pituitary LH content, and a decrease in testosterone/LH ratios at the highest dose. In female pups, although no effects were observed on plasma LH concentration, a similar significant elevation in pituitary LH content was observed during early puberty. Postpuberty, plasma LH and sex steroid concentrations were unaffected at any dose in spite of continued lead exposure. No significant effects were observed on epididymal sperm count in male pups at 85 days of age. In female pups, estrus cycling was only significantly disrupted at the highest dose of lead. These data suggest that the reproductive axis is particularly sensitive to lead during specific developmental periods, resulting in delayed sexual maturation produced by suppression by sex steroid biosynthesis. The mechanisms underlying this appear to involve lead actions on both LH release and gonadal function. Alt low, environmentally relevant blood lead concentrations, adaptation to the continuous presence of the metal ion occurs and surprisingly little effect is observed on adult reproductive endocrinology and physiology.