[phthalato(2-)]dioxotrilead

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:

Previous studies in Ronis' Lab (Ronis et al 1996) and by others (Sokol, 1990; Klein et all 1994) demonstrated that this level of lead acetate in drinking water produces reproductive and growth effects without compromising the overall health of the animals.

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

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: AAALAC-approved facility
- Diet (e.g. ad libitum): Ad libitum access to laboratory chow
- Water (e.g. ad libitum): Ad libitum access to drinking water
- Acclimation period: Rats were allowed to acclimatize to their new environment for 2 days prior to initiation of lead treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Constant temperature of 24 degrees Centigrade
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): Lights on between 6:00AM and 1800PM.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

lead acetate at 0.6% (w/v) (n=9) or an acetic acid solution containing an equivalent amount of acetate (n=8), beginning at gestation day 5. One milliliter of 5N HCL was added to each litter of each solution to preclude precipitaion of insoluble lead salts.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Previous studies in Ronis' Lab (Ronis et al 1996)and by others (Sokol, 1990; Klein et all 1994) demonstrated that this level of lead acetate in drinking water produces reproductive and growth effects without compromising the overall health of the animals.

Duration of treatment / exposure:

Groups of time-impregnated female Sprague-Dawley rats had ad libitum access to standard rat feed and either distilled deionized water (n=5), lead acetate at 0..6% (w/v) (n=9), or an acetic acid solution containing an equivalent amount of acetate (n=8), beginning at gestational day 5. One 5 N HCL was added to each liter of each solution to preclude precipitation of insoluble lead salts. At birth, pups were pooled from each treatment group, sexed, culled to four male and four female pups per liter, and randomly reassigned to water-, acetate-, or lead acetate-exposed dams to negate any potential litter effects and to generate the following experimental groups
1. Naive controls (NC) were continuously exposed to distilled deionized water (n=3 litters).
2. Acetic acid controls (Ac/Ac) were continuously exposed to acetic acid in the drinking water (n=3 litters).
3. Rats were cross-fostered from lead-exposed dams to acetic acid-exposed dams and thus only exposed to lead during gestation (Preg) (n=2 litters).
4. Rats were cross-fostered from acetic acid-exposed to lead-exposed dams in which lead exposure of the dams was until weaning and the pups, thereafter exposed to acetic acid and thus only exposed to lead duting lactation (Lact) (n=2 litters).
5. Gestationally lead-exposed pups were randomly placed back onto lead-exposed dams with lead exposure continuing throughout lactation and then stopped, resulting in lead exposure during pregnancy and lactation (P+L)(n=2 litters).
6. Pups were cross-fostered from acetic acid-exposed to lead-exposed dams at birth, in which lead exposure of the dams was continued until weaning and thereafter lead exposure of the pups was continued at the same level until sacrifice (Postnatal) (n=2 litters).
7. Pups exposed during gestation were randonly placed back onto lead-exposed dams and lead exposure was continued until sacrifice as described previously (Ronis et al., 1996) (Pb/Pb) (n=2 litters).

No. of animals per sex per dose:

experimental groups contained an n of 7-13 pups.

Control animals:

yes

Details on study design:

All experimental groups of pups were euthanized at age 85 days. Since the authors randomized pups from many litters to produce the treatment groups just listed, the unit of analysis was pup. As a result of occasional mis-sexing at birth, experimental groups contained an n=7-13 pups. Animals were weighed every 5 days, age of eye opening and vaginal opening were measured, and estrus cycling was monitored in the female pups from age 60 days. Rats were euthanized by decapitation, and blood was collected for analysis of lead, testosterone, and 17B-estradiol. In addition the testis, ovaries, and male secondary sex organs were dissected and weighed. One testis was fixed in Zenker's fixative for histological examination following sectioning and hematoxylin and eosin (H&E) staining. Serum was stored at -20 degrees centigrade until analysis.

Examinations

Ovaries and uterine content:

The testis, ovaries and male secondary sex organs were dissected and weighed.

Statistics:

All data are presented as mean +/- SEM. For all postnatal parameters statistical comparisons were made against the Ac/Ac control group. No statistically significant differences were observed for any postnatal parameter between the NC and Ac/Ac control groupss. For body weights, organ weights, blood lead, and serum sex steroid levels, statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc analysis. In order to adjust for differences in body weight, organ weights as a percentage of body weight were compared in addition to comparing absolute organ weights. Fo eye and vaginal opening, statistical analysis was conducted using the Wilcoxon rank sum test. (Hollander & Wolfe 1987) using a Bonferroni correction. Growth velocity was estimated using a mixed-effect model with a compound symmetry covariance structure that allowed for heterogeneity of this structure across gender. Models allowing for both gender and treatment effects on slope were estimated. All models allowed different intercept values for each gender-treatment combination. No statistical comparison of intercept was conducted, since these were expected to differ. Student's t-test were used to compare slopes between different treament groups within gender-age exposure categories. Separate models were fit, based on developmental stages: 5-20 days (lactation), 30-62 days (puberty), and 67-82 days (postpuberty). The most parsomonius model considering treatment and gender effects for each age category was determined by examination of the difference in -2 log likelihood value between nested models..

Indices:

lead analyses, organ weightsweight, estrus cycling, eye opening, vaginal opening, histological examination.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Remarks on result:

other: Study on offspring only

Maternal abnormalities

Abnormalities:

not examined

Description (incidence and severity):

Study on offspring only

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:

conc. level: Developmental toxicity

Effect level:

ca. 0.6 other: % (w/v)

Based on:

other: dissolved

Sex:

not specified

Basis for effect level:

other: See 'Remarks'

Remarks on result:

other: See 'Remarks':

Remarks:

Lead toxicity was most severe in continuously exposed animals, and the data suggests that the majority of effects of lead on reproduction and growth are direct effects at peripheral and/or central sites.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

The relative weights of male secondary sex organs in adult offspring were not significantly affected in any of the lead-treated groups. In contrast, female pups exposed to lead form birth through adulthood or from gestational day 5 through adulthood were observed to have significantly delayed vaginal opening and disrupted estrus cycling. These effects on female reproductive physiology were not observed in animals where lead exposure was confined only to pregnancy and lactation. Significant suppression of adult mean serum testosterone levels was only observed in male pups exposed to lead continuously from gestational age 5 days throughout life. Lead decreased birth weight in all animals exposed in utero and mean body weights were significantly decreased in all lead treated groups up to weaning. Analysis of growth curves revealed that all lead-treated groups had significantly reduced growth rates during lactation. However, in addition, male pups exposed to lead during pregnancy and lactation, from birth or gestational age 5 days, growth rates were also significantly reduced during puberty. Postpubertal growth rates were unaffected in any lead-treated group. Thus, delayed female reproductive development and suppression of adult male serum testosterone concentration required continuous exposure to the heavy metal. Little evidence was observed for an alteration of "endocrine imprinting" by lead on either reproductive or growth parameters. Exposure during development (pregnancy and lactation) resulted in no permanent effects in this model other than small (10%) decreases in the body weight of pups postpuberty.

Effects of lead on Vaginal Opening and Estru Cycling in Female Offspring

Treatment Group	Age (d) at 50% vaginal opening *	Cycling (%)**
NC (13)	36	100
Ac/Ac (12)	37	100
Preg (8)	37	89
Lact (9)	38	100
P + L (8)	37	89
Postnatal (8)	42 ***	50
Pb/Pb (9)	48***	11
Note:Number of pups/Treatment group given in parentheses*Age at which 50% of female pups were observed to have undergone vaginal opening**Percentage of female offspring cycling normally as assessed by microscopic analysis of vaginal smears taken during three cycles beginning at age 60 days. Data from n=8 -12 pups/treatment group.***Significant compared to Ac/Ac group based on Wilcoxson rank sum test of vaginal opening curves using Bonferroni correction of .007.

Effects of gestational lead exposure on offspring

		Treatment Group*	Births**	Pups***	M/F****	Dead*****	Body Weight******
		NC	5/5	10.4 =/- 1.5	29/22	3.8%	11.7 +/- 0.2
		Ac/Ac	8/8	13.0 +/- 0.7	56/47	2.3%	10.4 +/- 0.24*******
		Pb/Pb	6/9	10.5 +/- 1.0	23/29	19.0%*,**	7.6 +/- 0.25*******,********
		*NC, dams receiving distilled deionized water; Ac/Ac, dams receiving acetic acid solution; Pb/Pb, dams receiving 0.6% lead acetate (w/v)**Dams giving birth/impregnated animals***Number of pups/litter, mean +/-SEM****Number of male pups/number of female pups*****Stillbirths******Mean +/- SE, body weight at age 5 d (g)*******Significnat at p <.05 compared to NC********Significant at p < .05 compared to Ac/Ac				23/29  19.0%*,**  7.6 +/- 0.25*******,********         *nc, DA

Positive Effects of lead on male secondary sex organ weights in offspring at age 85 days

Treatment Group	Testis	Epididymis
Preg (8)	3.14 +/- 0.13* (0.94 +/- 0.02	0.96 +/- 0.03* (0.29 +/- 0.01)
P + L (8)	3.20 +/- 0.10 * (0.94 +/- 0.02)
Postnatal (8)		0.96 +/- 0.03 * (0.32 +/- 0.02)
Pb/Pb (7)	3.02 +/- 0.11 * (1.01 +/- 0.04)	0.86 +/- 0.02* 90.29 +/- 0.01)

Note: Number of pups/treatment group shown in parentheses in treatment group column. Data are absolute weights presnted as mean +/- SE. Data in parentheses are mean +/- SE percentge body weight.*Significant at p < .05 compared to Ac/Ac groupPositive Effects of lead on serum concentrations of testoserone and 17B-estradiol in offspring at age 85 days Treatment  Serum testosterone (Males)*   Pb/Pb  1.30 +/- 0.53** (7)  Note: Number of pups/treatment group given in parentheses *Data presented as mean +/- SE (pg/ml) ** Significant at p < .05 compared to Ac/Ac groups Statistical Analysis of growth curves in lead-exposed male and female offspring Treatment   Developmental period*   Male: Body weights (g)   Male: Slope *,**   Female: Body weights (g)***, ****   Female: Slope**,***  NC (m:11;F:13)  Weaning**** 50 +/- 1  2.33 +/- 0.06*******  44 +/- 1  2.30 +/- 0.05    Puberty *****  317 +/- 5  7.19 +/- 0.13  195 +/-5  3.28 +/_0.12    Postpuberty ******  384 +/- 6  2.53 +/_ 0.04********  223 +/- 6  1.43 +/- 0.04 *********  Ac/Ac (M:11.F:11)  Weaning  51 +/- 2  2.57 +/- 0.06  45 +/- 1  2.38 +/- 0.06     Puberty  314 +/-7  6.99 +/- 0.13  193 +/- 4  3.14 +/-0.13    Sacrifice(M)/Postpuberty (F)  379 +/- 9  2.53 +/- 0.04  224 +/-5  1.39 +/-0.01  Preg (M:8;F:*)  Weaning  38 +/-2*******  2.07 +/- 0.08*******  35 +/-1********  2.07 +/-0.07*******     Puberty   285 +/- 15  6.52 =/-0.18  177 +/-6 ********  3.42 +/-0.17    Postpuberty   348 +/- 20  2.52 +/- 0.06********  204 +/-6********  1.39 +/-0.01********  Lact (8)   Weaning   41 +/- 1*******  1.97 +/-0.07*******  35 +/-2********  1.92 +/- 0.07*******     Puberty   288 +/- 8*******  6.59 +/_0.15  182 +/-3  3.19 +/- 0.16     Postpuberty   361 +/- 8  2.53 +/_0.05********  220 +/- 4  1.38 +/-0.01********  P&L (8)   Weaning  39 +/-1*******  1.73 +/- 0.07*******  35 +/- 2********  2.02 +/- 0.07*******     Puberty  275 +/- 11*******  6.44 +/-0.16*******  183 +/- 3 3.48 +/- 0.17      Postpuberty  341 +/- 15*******  2.50 +/-0.04********  216 +/- 4  1.39 +/- 0.14********  Postnatal (8)   Weaning  42 +/- 15*******  1.98 +/-0.07*******  38 +/-1********  2.03 +/- ).07 *******     Puberty  247 +/-5*******  5.70 +/-0.15*******  180 +/- 5********  3.54 +/- 0.16     Postpuberty  307 +/-5*******  2.50 +/-0.04********  212 +/-5  1.30 +/- 0.12********  Pb/Pb (7)   Weaning  36 +/- 1*******  1.93 +/-0.07*******  33 +/- 1********  1.66 +/- 0.06*******     Puberty  232 +/- 5*******  5.56 +/- ).17********  163 +/-3********  3.32 +/- 0.15     Postpuberty  300 +/- 10*******  2.56 +/-).04*********  202 +/- 4********  1.26 +/- 0.07******** Note: Number of pups/treatment group given in parenthesis *Data analyzed as described in methods growth curves split into three linear sections representing lactation (5 -20 days), puberty (age 3 0 -62 days) and postpuberty (age 67 -83 days). **Slope of growth curve. Mean +/- SE for body weight gain, g/d ***Statistical analysis conducted using two-tailed p value comparing slope with that from the Ac/Ac group in the same time period ****Mean +/- SE for body weight at age 20 days; slope of growth curve age 5 -20 days *****Mean +/- SE for body weight at age 62 days; slope of growth curve age 30 -62 days ****** Mean +/- SE for body weight at age 85 days; slope of growth curve age 67 -82 days *******Significantly different at p<.05 from Ac/Ac reference category ********For this time period there was a significant gender effect at p<.05, but no significant difference in slopes by treatment

Applicant's summary and conclusion

Conclusions:

The effects of lead exposure during a number of developmental periods on reproductive physiology, endocrinology, and growth in the rat were studied. No evidence was found for permanent effects on th hypothalamic-pituitary-gonadal axis or on the male pubertal growth spurt following prenatal, postnatal, or perinatal lead exposure. This may be as a result of differences in the current study design compared to the prenatal exposure regimen of McGivern et al (1991). Extensive interlitter variability in lead effects might mask relatively small subtle imprinting effects. Alternatively, adaptation to the disorganizational effects of lead exposure in sexual differentiation may occur if the metal ion is continuously present from early embyonic development. This may involve mechanisms similar to the adaptation in the face of continued lead exposure that appears to occur in the reproductive and growth axis following puberty. Such an adaptive response to the effects of lead has been described in vitro in rat glioma cell cultures exposed to the metal (Dolzhanskaya et al., 1996). Lead toxicity was most severe in continuously exposed animals, and our data are suggestive that the majority of effects of lead on reproduction and growth are direct effects at peripheral and/or central sites.

Executive summary:

The reproductive, endocrine, and growth effects of developmental lead exposure were assessed using a rat model in which 0.6% lead acetate (w/v) was administered in the drinking water ad libitum during different developmental periods to determine if lead actions were a result of direct effects of continuous exposure to the metal ion or secondary to disrupted neonatal "endocrine imprinting." Sprague Dawley rats were exposed to lead: (1) from gestational day 5 through birth; (2) during pregnancy and lactation; (3) during lactation only ; (4) from birth through adulthood; (5) from gestational day 5 through adulthood. lead effects were measured on the development of aspects of the reproductive system, adult sex steroid levels, and growth rates, in both male and female animals.

The relative weights of male secondary sex organs in adult offspring were not significantly affected in any of the lead-treated groups. In contrast, female pups exposed to lead form birth through adulthood or from gestational day 5 through adulthood were observed to have significantly delayed vaginal opening and disrupted estrus cycling. These effects on female reproductive physiology were not observed in animals where lead exposure was confined only to pregnancy and lactation. Significant suppression of adult mean serum testosterone levels was only observed in male pups exposed to lead continuously from gestational age 5 days throughout life. Lead decreased birth weight in all animals exposed in utero and mean body weights were significantly decreased in all lead treated groups up to weaning. Analysis of growth curves revealed that all lead-treated groups had significantly reduced growth rates during lactation. However, in addition, male pups exposed to lead during pregnancy and lactation, from birth or gestational age 5 days, growth rates were also significantly reduced during puberty. Postpubertal growth rates were unaffected in any lead-treated group. Thus, delayed female reproductive development and suppression of adult male serum testosterone concentration required continuous exposure to the heavy metal. Little evidence was observed for an alteration of "endocrine imprinting" by lead on either reproductive or growth parameters. Exposure during development (pregnancy and lactation) resulted in no permanent effects in this model other than small (10%) decreases in the body weight of pups postpuberty.