Bis(D-gluconato-O1,O2)zinc

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

1

Reference substance name:

Zinc chloride

EC Number:

231-592-0

EC Name:

Zinc chloride

Cas Number:

7646-85-7

Molecular formula:

Cl2Zn

IUPAC Name:

Zinc chloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name of test material (as cited in study report): Zinc chloride

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley Breeding Laboratories (Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA).
- Age at study initiation: 30-35 days
- Housing: Groupedhoused (two animals of the same sex/cage) in polycarbonate cages with stainless-steel wire lids
- Diet: Rodent chow(Lab Diet, Richmond Standard, PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: Deionized water, ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature: 21.1 to 25.5 °C
- Humidity: 50-55%
- Air changes: 1/10 min
- Photoperiod : 12 h light/12 h dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Ninety seven percent ZnCl2 was dissolved in milli-Qwater and purity of the compound was greater than 97.00% (Fisher Scientific).

Details on mating procedure:

- Length of cohabitation: 21 d
- Proof of pregnancy: Conception (day 0 of gestation)was checked daily in the mornings by looking for the presence or absence of copulatory plugs.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 generations

Frequency of treatment:

7 days/week

Details on study schedule:

Dosing (7 days/week) started after two weeks of acclimation and was continued for males and females for 77 days prior to cohabitation. Dosing was continued throughout the periods of cohabitation (21 days) for both sexes. Dosing of female rats was continued throughout the gestation (21 days) and lactation (21 days) periods. The doses for both sexes were adjusted weekly according to changes in body weight.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were derived from a 14-day dose range finding study. The maximum tolerated dose (MTD) of ZnCl2 was set at 60 mg/kg/day in rats. In order to prevent a large effect of zinc-induced toxicity on non-reproductive tissues interfering with the interpretation of pure reproductive toxicity, the high-dose group (group 4) was set at 1/2 (30.00 mg of ZnCl2/kg bw/d) of the established MTD. Likewise, the middose group (group 3) was at 1/4 (15.00 mg of ZnCl2/kg of bw/d) of the established MTD and the lowest dose group (group 2) was 1/8 (7.50 mg of ZnCl2/kg bw/d) of the established MTD.
- Rationale for animal assignment: random

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

OTHER:
Hematology and clinical chemistry: Prior to necropsy, the Fo males were anesthetized with a combination of intraperitoneal Pentothal and Isoflo via inhalation. While the male rats were still under anesthesia, blood samples for hematology and clinical chemistries were collected in heparinised 3mL syringes via cardiac puncture. Following sample collection and while still under anesthesia, the animals were exsanguinated and necropsied. All plasma samples were analysed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), amylase (Amyl), blood urea nitrogen (BUN), creatinine (Crea), cholesterol (Chol), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (Phos), albumin (ALB), total protein (TP), total bilirubin (Tbil), and glucose (Glu) using Roche Cobas Mira S Chemistry Analyser (Roche Diagnostic System, Inc., Somerville, NJ).

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Not specified

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4sex/litter); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Total litter size, number of stillborn pups per sex, sex distribution, pup body weight and the presence of any obvious external congenital anomalies

GROSS EXAMINATION OF DEAD PUPS:
No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals, after the last litter of each generation were weaned

HISTOPATHOLOGY / ORGAN WEIGHTS:
Organ weights: During the necropsy, organ weights were recorded for the kidneys, liver, brain, pituitary, adrenals, pancreas, thymus, spleen, testes, epididymides, prostate, and seminal vesicles. Fo male organ weights were also adjusted to body weight for statistical analysis.
Histopathology: Tissue samples collected from organs listed above for histopathologic evaluation were fixed in either Bouins solution (all reproductive tissues) or 10% neutral buffered formalin (all other tissues). After fixation, the tissue samples were trimmed, processed, embedded in paraffin, cut at 6 μm and stained with hematoxylin and eosin.

Postmortem examinations (offspring):

At the end of cohabitation for the parental F1 males and lactation for the F1 females, the animals were anesthesized, sacrificed and their organ weights
were recorded like their Fo parents.

Statistics:

- Kruskal-Wallis test followed by the Mann-Whitney U test for pair-wise comparisons to detect the difference between treatment group and control means
- ANOVA for analysing body-weight change, fertility, litter size, pups’ viability, pups’ body weight, postpartum dam weight and organ weight data between different treatment groups
- Dunnett’s and/or Duncan’s multiple comparison procedures

Reproductive indices:

The reproductive parameters were expressed in terms of indices, weights, ratios and efficiencies that considered all stages from conception to weaning. The parameters were:
- Fertility index (%) = (number of females delivering/number of females cohabited) × 100
- Live birth index (%) = (number of live pups at Day 0/number of pups born) × 100
- 4-d survival index (%) = (number of live pups on Day 4/number of pups alive on day 0) × 100
- Body weights of pups = the body weight of pups were recorded on days 0, 4, 7, 14 and 21
- Sex ratio (%) = (the total number of males on the day of weaning)/ (the total number of females on the day of weaning) × 100
- Food efficiency = (body weight gain/amount of diet consumed) × 100

Offspring viability indices:

- 21-d (weaning) survival index (%) = (number of pups alive on Day 21/number of pups alive on Day 4) × 100
- Litter Size = Number of pups/number of pregnant females

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The most consistent finding was aggression/hyperactivity throughout the study in both males and females. Other less frequently seen observations noted are hair loss behind the ears in males, and vaginal discharges in low and high dose females.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The males experienced 0, 8, 20, and 12% mortality in control, low-, mid- and high-dose groups, respectively.
The mortality among the females was 12, 24, 28, and 24% for the control, low-, mid- and high-dose groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The ZnCl2-treated F0 males experienced significant reduction in body weight after the 1st week of dosing and this trend continued up to the end of the experiment. The total weight gain of males was significantly reduced (dose dependent) in the low-, mid- and high-dose groups.
In the F0 females, total weight gain and percent reduction in the low-, mid and high-dose groups were not significantly different from the control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

ZnCl2-treatment to F0 and F1 males and females caused no significant effects on their feed efficiencies when compared to their control groups.

Food efficiency:

no effects observed

Description (incidence and severity):

ZnCl2-treatment to F0 and F1 males and females caused no significant effects on their feed efficiencies when compared to their control groups.

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

None of the hemogram or leukogram values of both Fo and F1 males and females among the ZnCl2-treated groups were different from those of the control groups. However, there was a trend toward decreased values of Packed Cell Volume (PCV).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The clinical chemistry findings in males and females of both generations did not show any significant difference from those of their controls. However, in mid- and high-dose males of both generations, there seemed to a trend toward elevated values of Amyl, ALK, and GLu.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The most consistent finding was aggression/hyperactivity throughout the study in both males and females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In males, the most biologically meaningful lesions were found in the reproductive system (prostatic acinar atrophy and inflammation) and the hematopoietic-lymphoreticular system (splenic lymphoid depletion and hemosiderosis and thymic atrophy) of ZnCl2-treated groups. No significant changes in clinical pathology values or organ weights correlated with these lesions. None of the microscopic changes in target organs were of great magnitude. All unscheduled deaths were confined to the ZnCl2-treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the ZnCl2-treated females was similar to that seen in the males, except that no lesions were seen in the reproductive system. The correlations and biological interpretations were also very similar.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Postpartum dam body weight: The F0 and F1 post-partum dam weights in all dose groups were significantly different from their control groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

In F0 rats, ZnCl2 treatment caused a significant reduction on the fertility, litter size, and the viability indices (days 0 and 4) were significantly reduced at the high-dose group compared to control. However, no significant difference was seen in the weaning index and sex ratios F1 pups (Table 5). The body weight of F1 pups at day 21 in the high-dose group was significantly lower compared to their control.

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall effects for fertility and development toxicity. It is about 15 mg ZnCl2/kg bw/d, this corresponds to 7.2 mg Zinc/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The most consistent finding was aggression/hyperactivity throughout the study in both males and females. Other less frequently seen observations noted are hair loss behind the ears in males, and vaginal discharges in low and high dose females.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The males experienced 0, 12, 8, and 4% mortality in the control, low-, mid- and high-dose groups, respectively.
The mortality among the females was 0, 8, 12, and 20% in the control, low-, mid- and high-dose groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The F1 males in the mid- and high-dose groups experienced a significant reduction in body weight after the 1st week of dosing and the low-dose group experienced a similar reduction after the 2nd week of dosing. These trends continued up to the end of the experiment. The total weight gain of F1 males was significantly reduced (dose dependent) in the low, mid-, and high-dose groups.
The F1 females in the low-dose group experienced significant reductions in body-weight gain on weeks 15, 16, and 17. F1 females in the mid-dose group experienced a similar reduction in body-weight gain weeks 3 through 17. This was also true for females in the highest dose group for weeks 1 through 17.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

ZnCl2-treatment to F0 and F1 males and females caused no significant effects on their feed efficiencies when compared to their control groups.

Food efficiency:

no effects observed

Description (incidence and severity):

ZnCl2-treatment to F0 and F1 males and females caused no significant effects on their feed efficiencies when compared to their control groups.

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The clinical chemistry findings in males and females of both generations did not show any significant difference from those of their controls. However, in mid- and high-dose males of both generations, there seemed to a trend toward elevated values of Amyl, ALK, and GLu.

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In F1 males, the unadjusted weights of the brain, spleen, and prostate in all ZnCl2-treated groups, the liver, adrenal, testis and seminal vesicles in mid-dose and the kidney in high-dose were significantly different from their controls. When the organ weights of F1 males were adjusted for body weight, the brain, spleen, and prostate in all ZnCl2-treated groups, the liver, adrenal and seminal vesicles in mid-dose group, and kidney in high-dose group remained significantly different from their controls. The unadjusted organ weights of F1 females that were different from their controls included the brain and spleen in low- mid- and high-dose groups and the kidneys in the high-dose group. Following the adjustments of F1 female organ weights for body weight, the brain and spleen in all dose groups and kidneys in high dose groups were significantly different from controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross findings related to ZnCl2-treatment in males were primarily seen in the target organ systems (digestive, hematopoietic-lymphoreticular, and reproductive) already established for zinc. Digestive system lesions in the gastrointestinal tract (GIT) (distention, discoloration/hemorrhage and ulceration) and pancreas (smaller than usual) were mostly seen in rats given the two highest doses. Hematopoietic-lymphoreticular system lesions (small spleens and thymuses) were also scattered among the groups of ZnCl2-treated males. In the reproductive tract of the males, the only gross changes noted were small prostates and small seminal vesicles (one each) in the high-dose group. Gross lesions in ZnCl2-treated females generally paralleled those observed in their male counterparts.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, the most biologically meaningful lesions were found in the reproductive system (prostatic acinar atrophy and inflammation) and the hematopoietic-lymphoreticular system (splenic lymphoid depletion and hemosiderosis and thymic atrophy) of 30.00 mg/kg/day ZnCl2-treated groups. These results indicated that ZnCl2 exposure has only mild effects on the reproductive performance of rats. No significant changes in clinical pathology values or organ weights correlated with these lesions. None of the microscopic changes in target organs were of great magnitude. All unscheduled deaths were confined to the ZnCl2-treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the ZnCl2-treated females was similar to that seen in the males, except that no lesions were seen in the reproductive system. The correlations and biological interpretations were also very similar.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No significant difference was seen in the weaning index and sex ratios in F1 pups. In F1 generation rats, ZnCl2 treatment resulted in a significant reduction on fertility, viability (Day 0) and litter size in the high-dose group compared to control. However, ZnCl2 treatment showed no effect on viability index, weaning index and sex ratios of F2 pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The most consistent finding was aggression/hyperactivity throughout the study in both males and females.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Overall effects for fertility and development toxicity. It is about 15 mg ZnCl2/kg bw/d, this corresponds to 7.2 mg Zinc/kg bw/day.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

The possible adverse effects on reproductive performance observed are seen only at dose level causing marked systemic toxicity. Consequently they are not considered relevant for considering reproductive toxicity of the substance.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, administration of test material (ZnCl2) to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects (general toxicity) on adults and offspring at 30 and 15 mg/kg/d. Although effects were seen at 7.5 mg/kg/d, these were considered to be toxicologically non significant and is therefore considered to be the "No Observed Adverse Effect Level" (NOAEL). Considering possible reproductive effect, a NOAEL is fixed about 15 mg ZnCl2/kg bw/d.
The possible adverse effects on reproductive performance observed are seen only at dose level causing marked systemic toxicity. Consequently they are not considered relevant for classification purposes.

Executive summary:

A study was conducted to evaluate the reproductive toxicity potential of test material in rats for two generations.

Male and female rats were administered test material at the doses of 7.50, 15.00 and 30.00 mg/kg/d over two successive generations. Control group animals received deionised water. Exposure of F0 and F1 parental rats to test material showed significant reduction in fertility, viability (days 0 and 4), and the body weight of F1 and F2 pups from the high-dose group but caused no effects on litter size, weaning index, and sex ratio. Significant reduction in body weights of F0 and F1 parental males and postpartum dam weights female rats. Exposure of test material to F0 and F1 generation parental animals resulted in non-significant change in clinical pathology parameters (except the ALK level). Reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females was observed in F0 and F1 rats. Gross lesions were observed in gastro-intestinal (GI) tract, lymphoreticular/ hematopoietic and reproductive tract in parental rats in both generations. Reduced body fat was also recorded in F1 parental rats.

The possible adverse effects on reproductive performance observed are seen only at dose level causing marked systemic toxicity. Consequently they are not considered relevant for classification purposes.