Zinc nitrate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not applicable

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Limit test:

no

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 d
- Housing: 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 wk


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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 97% ZnCl2 was dissolved in milli-Q water.

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

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

2 generations

Frequency of treatment:

7 d/wk

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.

Dose / conc.:

7.5 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.

Positive control:

No data

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):

No data

Sperm parameters (parental animals):

No data

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

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Aggression/hyperactivity throughout the study in both males and females, hair loss behind the ears in males, vaginal discharges in low and high dose females; 0-20 and 12-24 % mortality in males and females respectively.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): All 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. The males experienced 0, 8, 20, and 12% mortality in control, low-, mid- and high-dose groups, respectively. 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.


HEMATOLOGY AND CLINICAL CHEMISTRY: 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). 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.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): 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.

ORGAN WEIGHTS (PARENTAL ANIMALS): In F0 males, the unadjusted weights of the brain in the midand high-dose groups, the liver and kidney in all ZnCl2-treated groups, the spleen in the high-dose group, and the seminal vesicles in the mid- and high-dose groups were significantly different from the control.When organ weights of F0 males were adjusted for body weight, the brain in the mid- and high-dose groups, the liver and kidney in all ZnCl2-treated groups, the spleen in the high-dose group, and the seminal vesicles in the mid- and high-dose groups remained significantly different from their controls. The unadjusted organ weights of F0 females revealed significant differences for the spleen and uterus in the high-dose group. Following the adjustments of F0 female organ weights for body weight, the spleen and the uterus in the high-dose group remained significantly different from their controls.


GROSS PATHOLOGY (PARENTAL ANIMALS): 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.


HISTOPATHOLOGY (PARENTAL ANIMALS): 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.


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

Dose descriptor:

LOAEL

Effect level:

7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING): 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 males experienced 0, 12, 8, and 4% mortality in the control, low-, mid- and high-dose groups, respectively. The mortality among the F1 females was 0, 8, 12, and 20% in the control, low-, mid- and high-dose groups, respectively.


CLINICAL SIGNS (OFFSPRING): Aggression/hyperactivity was observed throughout the study in both F1 males and females of ZnCl2-treated groups.


BODY WEIGHT (OFFSPRING): The body weights of F1 and F2 pups at Day 21 in the high-dose group were significantly lower compared to their control.


ORGAN WEIGHTS (OFFSPRING): 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 PATHOLOGY (OFFSPRING): 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.


HISTOPATHOLOGY (OFFSPRING): 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 FINDINGS (OFFSPRING): Reproductive performance: F1: 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.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

None

Conclusions:

Under the test conditions, administration of test material to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects 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).

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 Fo 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.

Under the test conditions, administration of test material to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects 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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In this dossier, the endpoint toxicity to reproduction is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all zinc substances of the zinc category. The assessment of the toxicity to reproduction of zinc and its substances is related to the assumption that once inorganic zinc compounds or zinc metal become bioavailable, this will be in the form of the divalent zinc cation. Further assuming that the anion of such inorganic zinc compounds can be regarded as “inert” with regard to repeated dose toxicity, the subsequent discussion focuses on the zinc cation. Further information on the read-across approach are given in the report attached to IUCLID section 13.2.

 

Animal studies – effects on fertility

Oral
In the two-generation reproduction toxicity study (Khan et al. 2007, non-GLP), groups of male and female SD rats were treated with 3 different dose levels of ZnCl2 via drinking water in comparison to a control group, and selected animals of the F1 generation were exposed to the same dose levels as received by their parents. For the conduct of the study, regulatory guidelines were taken into consideration.
Groups of 25 male and 25 female Sprague-Dawley rats (30-35 days, Harlan Sprague-Dawley, Inc, Indianapolis, IN, USA) received either 7.5, 15 or 30 mg/kg bw/d ZnCl2 via their drinking water 7 days/week. A concurrent control group received deionised water only. Treatment started after 2 weeks of acclimatisation and continued for 77 days prior to cohabitation and during cohabitation (21 days) for both sex. Female rats were treated throughout gestation and lactation periods. Pregnant females were allowed to deliver naturally, and litter sizes were standardized on day 4 after birth (4 male/4 female). On day 21 of lactation all F0 females and one male and female F1 offspring per litter were sacrificed. 25 pairs of F1 offspring from each group were selected to become F1 parents and were treated according to the same regime as their parents and mated to produce F2 offspring. F1 females were sacrificed at the end of the lactation period. Parental males of both generations were sacrificed after cohabitation.
The parameters evaluated in this multi-generation reproduction study reflect those recommended by the guidelines for generational toxicity studies.
Aggression and hyperactivity as well as hair loss behind the ears was observed throughout the study in both F0 and F1 parental male and females of the treatment groups. Vaginal discharge was seen in some females of the low and high dose groups. No effects of treatment were seen on the hemogram and leukograms of F0 and F1 animals of the ZnCl2 groups. There was a trend towards decreased values of packed cell volume. Clinical chemistry findings did not show significant differences from those of the controls. In mid and high dose males of both generations a trend towards elevated values of amylase, alkaline phosphatase and glucose were observed. The total weight gain of ZnCl2-treated males of both generations showed a significant reduction in all treatment groups compared to their controls. The mortality rates of male rats were 0, 8, 20 and 12% in the control, low, mid and high dose F1 males, and 0, 12, 8 and 4% in F2 males, respectively. However, the total weight gain of females of both generations did not show any significant difference. The post partum dam weight of the F0 and F1 rats were significantly different in all treatment groups compared to the control groups. The mortality rates were 12, 24, 28 and 24% in F0 females and 0, 8, 12 and 20% in F1 females, respectively. The absolute and relative kidney, liver, spleen, seminal vesicles weights of F0 males, and kidney, liver, spleen, adrenal, testis, prostate and seminal vesicle weights of F1 males were significantly reduced at 15 an 30 mg/kg bw/day ZnCl2. The absolute and relative spleen and uterus weights of F0 females and brain, kidney and spleen weights of F1 females were significantly reduced at 15 and 30 mg/kg bw/day during pre-mating, mating, gestation and lactation periods. Only minimal effects on reproductive performance were observed. ZnCl2 exposure caused significant reduction in fertility of both generations, F1 pup viability (day 0 and 4) and litter size in F2 offspring in the high dose group (30 mg/kg bw/d). No significant differences were seen in in weaning index and sex ratios of F1 and F2 pups. There was a clear effect of treatment on pups of both generations. Body weight of F1 and F2 pups on day 21 was significantly reduced in the high dose group compared to controls. Histopathological evaluations of tissues from rats of both generations revealed prostatic acinar atrophy and inflammation in the reproductive system, and the hematopoietic-lymphoreticular system (splenic lymphoid depletion and hemosiderosis and thymic atrophy) in the rats treated with 30 mg/kg bw/d.
In this multigeneration reproduction toxicity study in male and female SD rats treated over two generations, exposure of ZnCl2 resulted in adverse effects on fertility and viability and day 21 body weights in pubs of both generations at 30 mg/kg bw/d. Weaning index and sex ratios were not affected. Exposure to ZnCl2 via drinking water did not result in an effect on parental F0 and F1 female overall body weight gains, but post partum body weights were significantly reduced in both generations. In addition, body weights of males of the treatment groups were significantly reduced in F1 and F2 animals in comparison to control animals.
Based on the results presented in this publication, the lowest dose level of 7.5 mg/kg bw/day represents a LOAEL for parental toxicity. With respect to reproduction toxicity, only the high dose group treated with 30 mg/kg bw/d was affected and this dose level represents the LOAEL. Thus, the NOAEL for reproduction toxicity can be considered 15 mg/kg bw/d under the conditions of this study.
This multi-generation reproduction toxicity study was not conducted according to a regulatory guideline and not under GLP, however methods described follow general recommendations of official guidelines for reproduction toxicity and therefore, the study is considered acceptable and is thus reliable with restrictions [RL=2].

The study by Samanta and Pal (1986) was conducted to determine the effects of dietary zinc supplementation on male fertility in Charles-Foster rats. 4,000 ppm zinc as zinc sulphate was fed to 18 test males in diet for 30 -32 days. 15 control males were fed normal diet for the same duration. All animals mated with individual normal females once between Day 30 and 32. After mating, males were sacrificed for sperm characterization and zinc concentration analysis in different reproductive organs. Mated females were allowed to have full term gestation. Mating by treated males caused significant lowering of incidence of conception and number of live births per mated female. However, no stillbirth or malformed litter was observed. Motility of the sperm was significantly reduced in the treated rats but viability was unaffected. Zinc content was significantly increased only in the testis and sperm of the treated rats. The results indicate that dietary zinc supplementation at 4,000 ppm reduced male fertility in rats under the conditions of the study. Due to reporting and study design deficiencies, this study is considered not reliable (RL=3).

In the study by Talebi et al. 2013, the effect of zinc oxide nanoparticles on spermatogenesis was investigated. Groups of eight male NMRI mice received the substance in Milli-Q water via oral administration daily at dose levels of 5, 50 and 300 mg/kg bw/day for a duration of 35 consecutive days. A control group (distilled water) was run concurrently.
The results of the study demonstrated that zinc oxide nanoparticles induce testicular damage in a dose-dependent manner in mice. Epididymal sperm parameters including sperm number, motility and percentage of abnormality were significantly changed in 50 and 300 mg/kg bw/day zinc oxide nanoparticles treated mice. Histopathological criteria such as epithelial vacuolization, sloughing of germ and detachment were significantly increased in 50 and 300 mg/kg bw/day treated mice. 300 mg/kg bw/day zinc oxide nanoparticles induced formation of multinucleated giant cells in the germinal epithelium. 50 and 300 mg/kg bw /day zinc oxide nanoparticles also caused a significant decrease in seminiferous tubule diameter, seminiferous epithelium height and maturation arrest. The presence of vacuoles in the cytoplasm of Sertoli cells shows direct damage to these cells. The multinucleated giant cell formation and sloughing of immature germ cells from the seminiferous tubules indicates that these nanoparticles might also affect Sertoli cell functions.
In conclusion, evidence of testicular damage was observed at dose levels of 50 and 300 mg/kg bw/day in male NMRI mice, while the lowest dose level of 5 mg/kg bw/d represents a NOAEL with only very minimal findings.
The publication shows limitations with respect to presentation and reporting of data. Firstly, the test substance was described insufficiently (purity and stability missing). Furthermore, the number of animals is too low, which significantly reduces the statistical power and reduces the meaningful evaluation of the effect of the test item. In addition, the authors did not state the type of oral administration (e.g. oral gavage) and no information was given about the determination of the actual concentration. The missing information on actual concentration makes it impossible to determine, if the animals received the concentrations as claimed by the authors. The dose level chosen for the high dose group seems somewhat implausible because it is known from other studies that 300 mg/kg bw/day is clearly toxic to mice, but no general findings of toxicity were reported (clinical signs, mortality, body weight, gross pathology and histopathology (exception: testes) not reported). The authors investigated sperm parameters in the current study, however not enough spermatozoa were examined for sperm morphology, which limited the evaluation for test item related effects on morphology. Also, epididymis weight was not determined during the study. Lastly, individual data and historical control data were not presented, which makes it impossible to observed outliners. Based on the shortcomings, as described above, the study is regarded as not reliable and only of supportive nature.

Bara et al. (2018) investigated the effect of zinc oxide nanoparticles on male and female reproductive organs (female mice corpora lutea and male offspring testes) in comparison to bulk zinc oxide. Groups of four pregnant Swiss albino females were exposed to either zinc oxide nanoparticles or zinc oxide (bulk) via gavage for 2 days on alternate days during gestation days 15 -19. Animals were treated either with vehicle (distilled water) or 50, 100 and 300 mg/kg bw zinc oxide nanoparticles or 100 mg/kg bw bulk zinc oxide.
It could be demonstrated that prenatal exposure to zinc oxide nanoparticles altered the steroidogenesis-related gene expression in the testis of male mice, but no change in the serum testosterone concentration was recorded in the zinc oxide nanoparticle-exposed male mice, while bulk zinc oxide exposure had increased the serum testosterone concentration. Increased testicular weight were observed in the 100 mg/kg bw of zinc oxide nanoparticle-treated group. Pathological changes like prominent epithelial vacuolization, decreased seminiferous tubule diameter and low intracellular adhesion of seminiferous epithelia were observed in the testis of prenatally exposed male mice with 50 or 100 mg/kg bw zinc oxide nanoparticles. Oral intake of 300 mg/kg zinc oxide nanoparticles by pregnant mice was shown to be lethal to the developing foetus. Miscarriages were observed in the pregnant mice treated with only a single dose of 300 mg/kg bw of zinc oxide nanoparticles. No change in the histological sections of placenta was observed in the exposed mice compared with the control indicating that oral gavage of zinc oxide nanoparticles (50 or 100 mg/kg bw) did not cause in situ placental damage. A decrease in StAR and no change in the P450scc, 3ßHSD and LHr gene expression were observed in corpora lutea of 100 mg/kg bw of zinc oxide nanoparticle-exposed mice. In testis, a marked increase in the relative expression of the StAR gene after in utero exposure to zinc oxide nanoparticles, bulk zinc oxide was observed. While increased P450scc in the lower zinc oxide nanoparticles concentration (50 mg/kg bw) or bulk zinc oxide-exposed groups was observed, no change in the other steroidogenesis related genes (P450c17, LHr or 17ßHSD) was found. The results indicate that in utero exposure of ZnO NPs or bulk ZnO may interfere with the steroidogenesis, mainly by affecting the StAR or P450scc gene expression levels. However, no linear relationship was observed between the StAR gene expression and testosterone production.
In conclusion, the data suggest that oral exposure of zinc oxide nanoparticles in mice during pregnancy affects steroidogenesis in the corpora lutea of pregnant mice (via progesterone biosynthesis) and the testis of male offspring (via testosterone biosynthesis) exposed in utero. It could be shown that foetal nanoparticle exposure of zinc oxide (50 and 100 mg/kg bw) may disturb the reproductive functions of males by evidenced from gross pathological changes in testis.
The publication shows limitations with respect to presentation and reporting of data. Firstly, the test item was insufficiently described. In addition, the study was conducted only in a very small number of animals and animals were treated only on 2 days at the end of the gestation period and shows thus significant methodological deficiencies. The actual concentration and stability of the dosing solutions were not verified. Although the relevant route of exposure (oral) was chosen, the data can be regarded only as supportive and are not reliable (RL=3).

 

non-physiological routes of exposure
In the present study, male Wistar rats were exposed daily to ZnO nanoparticles at dose levels of 50, 100, 150, and 200 mg/kg bw/day for a duration of 10 days via intraperitoneal injection (Abbasalipourkabir et al. 2015). A control group (bi-distilled water) was run concurrently.
Significant changes were observed in oxidative stress parameters. In the 50 - 200 mg/kg bw/day groups, the malondialdehyde (MDA) levels and aspartate aminotransferase (AST) activity were significantly increased, and alanine aminotransferase (ALT) activity were significantly increased in the 150 and 200 mg/kg bw/day group compared to the controls. A significant decrease in the total antioxidant capacity (TAC) and significant increase in the total oxidant status (TOS) were observed in the 200 mg/kg bw/day group compared to the control group. However, no significant changes in superoxide dismutase or glutathione peroxidase (GPX) activity were observed in the 50 – 200 mg/kg bw/day groups compared to controls. There were pathological changes including the proliferation of glomerular cells, inflammation of interstitial tissue and congestion of glomerulus in kidney of all groups treated with ZnO NPs at concentrations above 50 mg/kg body weight. Liver tissue of animals exposed to ZnO NPs showed increased Kupffer cells, congestion, inflammation in the liver parenchymal, ballooning, port inflammation and chromatin condensation. The sperm quality on males was affected by ZnO NP exposure (ip) in a dose-dependent manner starting at the low dose level of 50 mg/kg bw.
The study appears to be appropriately conducted and well documented and therefore regarded as acceptable but only supportive, because the route of administration chosen (ip) is not guideline conform and not suitable to assess reproduction toxicity. The publication shows significant methodological deficiencies in the experimental setup and documentation (non GLP, too short exposure period of 10 day, only male animals). Dosing scheme of just 10 days is not justified. The actual concentration and stability of the dosing solutions were not verified. Number of animals per group (n=6) was too low for a statistical evaluation.
Toxicity on reproduction in male rats was not evaluated (only oxidative status, sperm analysis and histochemical analysis of the liver and kidney). Males were not mated following test item administration. Therefore, it is not possible to investigate the effect of the test item on the ability of the sperm to produce healthy and alive offspring, which makes it impossible to obtain further information about the effect on fertility by the test material. Clinical observations were not recorded. Animals were weighed before the study, but not throughout the study or at study termination. Food consumption were not recorded. No gross pathological examination was conducted. Organ weights were not recorded, and with exception of the liver and kidneys, the testes, epididymis, seminal vesicles, prostate, coagulating gland and pituitary gland were not examined histologically. Gross pathology and histopathology of the accessory sex organs would provide further information on fertility effects.
Histopathological findings observed have not been tabulated by dose level. Therefore, it is not possible to determine how the histopathological findings differ between the dose levels or compared to the control group, and whether these were dose-related. Number of animals examined per group was not specified in the results (see tables 2 and 3 with the biochemical parameters, or table 3 with data on sperm analysis). Additionally, information on the temperature, relative humidity and air changes for the environmental conditions of the test animals were missing.
In addition, the dose regimen appears grossly implausible, as mortality was seen after oral dosing with 300 mg/kg bw/day in other RDT studies. Since i.p. dosing may be assumed to increase the overall systemic availability, one would expect severe signs of toxicity in at least the high-dose animals. Therefore, the data from this mechanistic study are disregarded due to major methodological deficiencies and regarded as not reliable. Due to major methodological deficiencies, the data of this mechanistic study are disregarded and are not considered to be reliable.

In this mechanistic, non-guideline toxicity study (non-GLP), the effects of ZnO NP on male reproductive organs were evaluated in male CD-1 mice in a single dose toxicity study with intravenous administration (Han et al. 2016). Test material: ZnO NP (Beijing DK nanotechnology Co. Ltd (Beijing, People’s Republic of China). Twenty-one day old male mice (Vital River, Beijing, People’s Republic of China) were divided into three groups (more than six animals per group) and received either vehicle or 1.0 or 5.0 mg/kg bw/d ZnO NP by single intravenous tail injection. Testes (six testes from six mice from each group) were collected at PND28 and PND42 and processed for standard histological assessments. At PND49, the epididymis were dissected from control and treated mice and the sperm morphology of was examined.
ZnO NPs caused alteration in the structure of seminiferous epithelium and the production of morphologically abnormal spermatozoa. A significant reduction in the thickness of the seminiferous epithelium was observed after injection of males with 5 mg/kg bw/d ZnO NPs at PND28 and PND42. The diameter of the seminiferous tubules was also reduced in the ZnO NP-treated mice, although changes were slight in the group treated with 1 mg/kg ZnO NPs. Moreover, the percentage of spermatozoa showing an altered morphology (double head, small head, unshaped head, double tail) at 49 days after ZnO NP treatment was significantly higher in comparison to controls (P<0.05 or P<0.01).
This mechanistic toxicity study in male mice supports the evidence of an effect of exposure to ZnO NPs on male reproductive organ morphology and sperm quality. Materials and methods are described only briefly, and results are presented only as summary without details. The route of exposure chosen (iv) does not represent a relevant route in the context of risk assessment and single administration is not appropriate for the assessment of this endpoint. Therefore, the study is only of supportive nature and regarded as not reliable [RL=3]

This non-regulatory study (Zhai et al. 2018) represents a mechanistic study (non-GLP) to evaluate the effects of maternal ZnO NPs exposure on female reproductive organ development of offspring in vivo and in vitro. Test material: ZnO NP (Beijing Sorlabio Life Science Co. LTD (YZ-111619, Beijing, China)), Average diameter: 30 nm, Purity: 99.9%, Specific surface area: 50 cm2 /g, bulk ZnO (Beijing Sorlabio Life Science Co. LTD (YZ-111619, Beijing, China)), Average diameter: 230 nm, ZnSO4 (Sigma-Aldrich, Inc (Sigma Z0251, USA))
It was studied whether maternal exposure to nZnO during embryo development affects oocyte DNA integrity and the establishment of the ovarian reserve in female offspring by using an in vitro ovary culture system. Pregnant CD1 mice from Beijing Vital River Laboratory Experimental Animal Technology Co. LTD (Beijing, China) were used. Ovaries were isolated from 12.5 dpc mouse embryos and cultured in the presence of nZnO for 6 days. In addition, 16 mg/kg body weight ZnO NP was injected intravenously on 12.5 dpc in pregnant mice on two consecutive days and the ovaries of foetuses or offspring were analysed at three critical periods of oogenesis: 17.5 dpc, 3 days post‐partum (dpp) and 21 dpp.
Germinal vesicle (GV)-intact oocytes were isolated from 4 – 6 week female mice and observed under a fluorescence microscope. Foetal oocyte cytospreads were performed and evaluated under a fluorescence microscope. Ovaries were processed for immunostaining and immunohistochemistry. TUNEL assay, transmission electron microscope (TEM) analysis, western blotting and quantitative real-time PCR (qRT-PCR) was conducted with ovarien tissue.
In order to investigate whether exposure to ZnO NP during embryonic development affects ovarian development, 12.5 day post coitum (dpc) foetal mouse ovaries were cultured in the presence of ZnO NP for 6 days. The nanoparticles (NPs) accumulated within the oocyte cytoplasm in a dose dependent manner, caused DNA damage and apoptosis, and result in a significant decrease in oocyte numbers. No such effects were observed when the ovaries were incubated in the presence of ZnSO4 or bulk ZnO as controls. In addition, intravenous injection of 16 mg/kg body weight nZnO in 12.5 dpc pregnant mice revealed evidence of increased DNA damage in pachytene oocytes in foetal ovaries and impaired primordial follicle assembly and folliculogenesis dynamics in the ovaries of the offspring were found. The results indicate that certain types of NPs may affect pre‐ and post‐natal oogenesis in vitro and in vivo.
The mechanistic study is not relevant in a regulatory context and shows major deficiencies with respect to description of the methods and reporting. The treatment regimen of pregnant mice and number of animals used is not described, and also the sequence of subsequent methods used for assessment is difficult to extract. Thus, test system used is unsuitable and documentation is insufficient for assessment and therefore the study is regarded as not assignable [RL=4].

 

Conclusion
In a reliable (with restriction) multi-generation reproduction toxicity study (Khan et al. 2007), exposure of ZnCl2 resulted in adverse effects on fertility at 30 mg/kg bw/d in male and female SD rats treated over two generations with no effects on fertility at 15 mg/kg bw/d. The lowest dose level of 7.5 mg/kg bw/d was regarded as the LOAEL for parental toxicity.
Effects on male reproductive organs were already described by Edwards and Buckley (1995). It was shown that at 335 mg Zn/kg bw/day administered via the diet all males showed hypoplasia in testes and seminiferous tubules. However, these findings need to be seen in the context that these high dose group animals were generally of poor health conditions and killed for humane reasons prior to study termination. Rats exposed to 13 or 60 mg Zn/kg bw/day in the diet over a period of 90 days did not show any detrimental effects on sex organs.
Two mechanistic repeated dose toxicity study with evaluation of the effects of ZnO nanoparticles on male reproductive organs (Abbasalipourkabir et al. 2015, Han et al. 2016), and two additional non-regulatory toxicity studies, in which the effects on reproductive organ development of offspring from prenatally exposed dams were investigated (Bara et al. 2018, Zhai et al. 2018). Three of these studies (Abbasalipourkabir et al. 2015, Bara et al. 2018, Han et al. 2016) were conducted with a non-relevant route of exposure (ip or iv) and one study was regarded as not assignable (Zhai et al. 2018), because of the use of an unsuitable test system and insufficient documentation. Therefore, all studies are regarded as only of supportive nature and either not reliable or assignable [RL=3/4] in a regulatory context. In addition, data from one this reproductive and development toxicity screening study (Jo et al. 2013) conducted in consideration of OECD guideline 421 are available, however the study was conducted only with one high dose level and is thus also regarded as not reliable [RL=3].
Although conducted by an irrelevant route of exposure, the results of the 10-day short-term repeated dose toxicity study (Abbasalipourkabir et al. 2015) conducted by ip administration of ZnO NP (0, 50, 100, 150 and 200 mg/kg bw/d) in male Wistar rats support the evidence of an effect of zinc on male reproductive organs (sperm count, vitality, motility and morphology) from a mechanistic point of view. Oxidative stress induced by ZnO is discussed as the mechanistic basis for the observed findings at and above 50 mg/kg bw/d ZnO nanoparticles (LOAEL).
In addition, the mechanistic toxicity study with a single intravenous dose of ZnO NPs (1 mg/kg bw) in male mice supports the evidence of an effect of exposure to ZnO NPs on male reproductive organ morphology and sperm quality (Han et al. 2016).
The effects ZnO nanoparticles in comparison to bulk ZnO were investigated in a non-regulatory developmental toxicity study in pregnant albino mice (Bara et al. 2018) treated either with vehicle, 50, 100, 300 mg/kg bw ZnO NPs or 100 mg/kg bulk ZnO by oral gavage for 2 days on alternate days during gestation day (GD) 15–19. The effects of treatment on corpora lutea of dams was investigated after weaning and on the testis of male offspring on PND60. Gross pathological changes in testis of male mice and effects on sperm parameters were observed in animals receiving ZnO nanoparticles prenatally at and above 50 mg/kg bw in comparison to 100 mg/kg bw bulk ZnO, but serum testosterone concentrations were only increased in offspring of the ZnO bulk group. In addition, exposure to ZnO NPs altered the steroidogenesis-related gene expression in the testis of male mice and corpora lutea of dams.
No effect on male and female fertility and mating/gestation period was observed in a reproductive and development toxicity screening study in male and female SD rats (Jo et al. 2013). The number of implants was similar between groups, indicating that pregnancy rate, mating performance and post-implantation were unaffected by treatment with one high oral dose (500 mg/kg bw/d ZnO Nps) given by gavage to male (2 weeks before mating) and female rats (2 weeks before mating to postnatal day 4).
In a non-regulatory and not assignable toxicity study, the effects of ZnO NP on female reproductive organ development of foetuses was investigated in CD1 mice (Zhai et al. 2018) by intravenous administration of 16 mg/kg bw ZnO NP on two consecutive days on day 12.5 during pregnancy and evaluation of foetal ovaries. In addition, the effects on oocyte cultures of adult mice were investigated in vitro. In this study, evidence of increased DNA damage in pachytene oocytes in foetal ovaries and impaired primordial follicle assembly and folliculogenesis dynamics in the ovaries of the adult offspring were found.
The available information suggests that high oral doses of zinc (i.e., exposure levels greater than 20 mg Zn/kg bw/day) may adversely affect spermatogenesis and result in impaired fertility indicated by decreased number of implantation sites and increased number of resorptions (US EPA, 2005). However, these effects were only observed in the presence of maternal toxicity as seen in the one- or two-generation studies conducted by Khan et al. (2001, 2003, 2007) or, in case of the study conducted by Kumar et al. (1976), when other non-zinc relevant study specificities could have impacted the study outcome.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 -14 weeks
- Weight at study initiation: 146.0 - 192.1 g
- Fasting period before study: no
- Housing: individually, dust-free wooden bedding, enrichment: wooden gnawing blocks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%.
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose/head only

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: individual acrylic tubes
- Source and rate of air: conditioned and compressed air, 6 m3/h
- System of generating particulates/aerosols: feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 +/- 2°C, 50 +/- 20%,
- Air flow rate: 6 m3/h
- Method of particle size determination: cascade impactor/ Marple impactor
- Treatment of exhaust air: disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrically by filter samples
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres of test group 1 – 3 were analyzed by gravimetrical analysis. Daily means were calculated based on two measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In these groups, the constancy of concentration in each inhalation system was continuously monitored using scattered light photometers.
In the control group (test group 0) no sample was analyzed over the study period. The analyses were carried out at the Inhalation Laboratory of the Experimental Toxicology and Ecology of BASF SE.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”) and supplied on GD 0 (= detection of vaginal plug/sperm). The animals arrived on the same day (GD 0) at the experimental laboratory.

Duration of treatment / exposure:

6 hours on 14 consecutive days

Frequency of treatment:

daily

Duration of test:

from implantation to one day prior to the expected day of parturition

Dose / conc.:

0.3 mg/m³ air

Dose / conc.:

1.5 mg/m³ air

Dose / conc.:

7.5 mg/m³ air

No. of animals per sex per dose:

25 pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: request of the sponsor
- Rationale for animal assignment: random
The inhalation route was selected since it is relevant in terms of potential human exposure.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation day (GD) 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.
The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION
The consumption of food was recorded for GD 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day #20
- Organs examined: Organ weights of the lungs, the unopened uterus and the placentas were determined. For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated by resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external, soft tissue and skeletal (incl. cartilage) findings. Larynx, lungs, mediastinal and tracheobronchial lymph nodes, trachea and nasal cavities were examined histopathologically in the control and high-dose groups.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

clinical and fetal examination:
Simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means
Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Pathology:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians

Historical control data:

used to interprete results of present study

Dose descriptor:

NOAEC

Effect level:

1.5 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEC

Effect level:

7.5 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
no effects observed

Dose descriptor:

NOAEC

Effect level:

7.5 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

The following test substance-related adverse effects/findings were noted:

 

 

Test group 3 (7.5 mg/m³):

 

·        Increase in absolute and relative lung weights (up to 47%)

·        Moderate alveolar lipoproteinosis in all animals examined

·        Slight, diffuse inflammation in all animals examined

·        Minimal to slight focal hemorrhage in three of ten animals

·        No test substance-related adverse effects on gestational parameters or fetuses

 

 

Test group 2 (1.5 mg/m³):

 

·        No test substance-related adverse effects on dams, gestational parameters or fetuses

 

 

Test group 1 (0.3 mg/m³):

 

·        No test substance-related adverse effects on dams, gestational parameters or fetuses

 

Executive summary:

Under the conditions of this prenatal developmental toxicity study, the inhalative administration of Z-Cote HP1 to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 7.5 mg/m³ caused moderate alveolar lipoproteinosis and slight inflammation. These histopathologic findings are regarded to be adverse in nature. The relevance for humans however is not clear.

 

In conclusion, the no observed adverse effect concentration (NOAEC) for maternal toxicity is1.5 mg/m³.

 

The no observed adverse effect concentration (NOAEC) for prenatal developmental toxicity is7.5 mg/m³.There were no adverse fetal findings evident at any dose.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Used in risk assessment report for zinc sulphate, acceptable for assessment.

Principles of method if other than guideline:

Female hamsters were administered daily doses of 0.9, 4.1, 19 and 88 mg/kg bw by gavage during days 6-10 of gestation to evaluate the developmental toxicity of zinc sulphate.

GLP compliance:

not specified

Limit test:

no

Species:

hamster

Strain:

not specified

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

no

Details on mating procedure:

No data

Duration of treatment / exposure:

Days 6-10 of gestation

Frequency of treatment:

Daily

Duration of test:

No data

No. of animals per sex per dose:

23-25 animals/group

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

88 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

88 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

- No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed.

- The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment.

- No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Conclusions:

Under the conditions of the test, administration of up to 88 mg/kg bw of unspecified zinc sulphate (ca. 35.2 mg or 19.9 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult hamsters and their foetuses.

Executive summary:

Female hamsters (23-25 animals/group; outbred strain of golden hamster) received daily doses of 0.9, 4.1, 19 and 88 mg unspecified ZnSO4/kg bw by gavage during days 6-10 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 8, 10 and 14 of gestation. The females were sacrificed at day 14. The urogenital tract of each animal was examined in detail. Between 21 and 24 females were pregnant at term in all groups.

No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Under the conditions of the test, administration of up to 88 mg/kg bw of unspecified zinc sulphate (ca. 35.2 mg or 19.9 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult hamsters and their foetuses.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Used in risk assessment report for zinc sulphate, acceptable for assessment.

Principles of method if other than guideline:

Female CD-1 mice were administered daily doses of 0.3, 1.4, 6.5 and 30 mg ZnSO4/kg bw by gavage during days 6-15 of gestation to evaluate the developmental toxicity of zinc sulphate.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

No data

Duration of treatment / exposure:

Day 6-15 of gestation

Frequency of treatment:

Daily

Duration of test:

no data

No. of animals per sex per dose:

25-30 animals/group

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

- No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed.

- The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment.

- No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Conclusions:

Under the conditions of the test, administration of up to 30 mg/kg bw of ZnSO4 (ca.12 mg or 6.8 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult mice and their foetuses.

Executive summary:

Female CD-1 mice (25-30 animals/group) received daily doses of 0.3, 1.4, 6.5 and 30 mg unspecified ZnSO4/kg bw by gavage during days 6-15 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 6, 11, 15 and 17 of gestation. The females were sacrificed at day 17. The urogenital tract of each animal was examined in detail.

No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Under the conditions of the test,administration of up to 30 mg/kg bw of unspecified zinc sulphate (ca.12 mg or 6.8 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult mice and their foetuses.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Used in risk assessment report for zinc sulphate, acceptable for assessment.

Principles of method if other than guideline:

Female Dutch rabbits were administered daily doses of 0.6, 2.8, 13 and 60 mg/kg bw by gavage during days 6-18 of gestation to evaluate the developmental toxicity of zinc sulphate.

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

Dutch

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

no

Details on mating procedure:

No data

Duration of treatment / exposure:

Days 6-18 of gestation

Frequency of treatment:

Daily

Duration of test:

No data

No. of animals per sex per dose:

14-19 animals/group

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

- No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed.

- The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment.

- No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Conclusions:

Under the conditions of the test, administration of up to 60 mg/kg bw of unspecified zinc sulphate (ca. 24 mg or 13.6 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult rabbits and their foetuses.

Executive summary:

Female Dutch rabbits (14-19 animals/group) received daily doses of 0.6, 2.8, 13 and 60 mg unspecified ZnSO4/kg bw by gavage during days 6-18 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 6, 12, 18 and 29 of gestation. The urogenital tract of each animal was examined in detail. The females were sacrificed at day 29. Between 10 and 12 females were pregnant at term in all groups.

No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Under the conditions of the test, administration of up to 60 mg/kg bw of unspecified zinc sulphate (ca. 24 mg or 13.6 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult rabbits and their foetuses.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Used in risk assessment report for zinc sulphate, acceptable for assessment.

Principles of method if other than guideline:

Female Wistar rats were administered daily doses of 0.4, 2.0, 9.1 and 42.5 mg/kg bw by gavage during days 6-15 of gestation to evaluate the developmental toxicity of zinc sulphate.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

no

Details on mating procedure:

No data

Duration of treatment / exposure:

Days 6-15 of gestation

Frequency of treatment:

Daily

Duration of test:

No data

No. of animals per sex per dose:

25-28 animals/group

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

42.5 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

42.5 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

- No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed.

- The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment.

- No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Conclusions:

Under the conditions of the test, administration of up to 42.5 mg/kg bw of zinc sulphate (ca. 17 mg or 9.6 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult rats and their foetuses.

Executive summary:

Female Wistar rats (25-28 animals/group) received daily doses 0.4, 2.0, 9.1 and 42.5 mg unspecified ZnSO4/kg bw by gavage during days 6-15 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 6, 11, 15 and 20 of gestation. The females were sacrificed at day 20. The urogenital tract of each animal was examined in detail. At term 25 females were pregnant in all groups.

No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups.

Under the conditions of the test, administration of up to 42.5 mg/kg bw of zinc sulphate (ca. 17 mg or 9.6 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult rats and their foetuses.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Homogeneity of the test substance in the formulations which the animals received were not analysed. Clinical signs and gross pathological findings observed have not been tabulated by group. No historical control data are available. Pre- and postimplantation loss were determined, but the results were not shown. Gravid uterus was weighed, but it is unclear whether the cervix was included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

yes

Remarks:

No analysis of test material homogeneity in formulations; clinical signs have not been tabulated by group or sex; no historical control data; no results of pre- and post-implantation loss; unclear whether gravid uterus was weighed with cervix.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Source and Lot no. of test material: Sumitomo-Osaka Cement Co., Ltd., Tokyo, Japan; Lot no. 141319.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Inc., Gyeonggi-do, Korea
- Age at study initiation: 12 weeks old (10 weeks old at purchase + 12 days of quarantine and acclimation)
- Weight at gestation day 0 (mean values): 284.9 ± 13.83 g (control group); 287.7 ± 11.68 g (low-dose group); 286.8 ± 13.78 g (mid-dose group) and 285.5 ± 14.09 (high-dose group)
- Housing: mated females were housed individually in clear polycarbonate cages with stainless steel wire lids.
- Diet (ad libitum): commercial rodent chow, supplier: Cargill Agri Purina, Inc., Gyeonggi-do, Korea
- Water (ad libitum): sterilized tap water after UV irradiation
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.8 - 23.0 °C
- Relative humidity: 45.3 - 56.9 %
- Air changes: 10 - 15 air changes/hour
- Photoperiod: 12 /12 hrs dark / hrs light

Route of administration:

oral: gavage

Vehicle:

other: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer

Remarks:

with 2 % L-serine

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
NPs were suspended in 20 nM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer with 2 % L-serine and then mixed well. The final pH of the buffer solution was adjusted with hydrochloric acid to 6.5, and 20 % of the surface-modified ZnO(SM20(+)) NPs were used as a stock solution. Before the administration, the suspension was stirred for 10 seconds and then diluted with distilled water.
- Dosing volume: 10 mL/kg (the daily application volume was calculated in advance based on the most-recently recorded body weight of the individual animal.

VEHICLE:
- Justification for use and choice of vehicle: to cap ZnO NPs with L-serine molecules, organic ligands, which are widely used capping agents for inorganic NPs to enhance the surface charge property (Degen, A. et al. (2000), Oh, J.M. et al. (2011) and Kim, K.M. et al. (2012)).*

*References:
- Degen, A., Kosec, M. Effect of pH and impurities on the surface charge of zinc oxide in aqueous solution. J Eur. Ceram. Soc. 2000; 20: 667-673.
- Oh, J.M., Park, D.H., Choy, J.H. Integrated bio-inorganic hybrid systems for nano-forensics. Chem. Soc. Rev. 2011; 40(2): 583-595.
- Kim, K.-M., Kim, T.-H., Kim, H.-M., et al. Colloidal Behaviors of ZnO Nanoparticles in Various Aqueous Media. Toxicol. Environ. Health Sci. 2012; 4(2): 121-131.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of dosing solution was measured on gestational day 5, 11, and 19 using inductive coupled plasma atomic emission spectrometry (ICP-AES).
Results:
Concentrations: three analyses confirmed that the analysed concentrations of all dose formulations were within ±15 % of the target concentrations. Concentra¬tions of total zinc were 9.82 ± 0.84 mg/mL for the 100-mg/kg/day group, 20.04 ± 1.63 mg/mL for the 200-mg/kg/day group, and 40.80 ± 1.47 mg/mL for the 400-mg/kg/day group.
Stability: ZnO(SM20(+)) NPs were stable for 4 hours at room temperature.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male / 1 female
- Proof of pregnancy: each morning, the female rats were examined for the presence of sperm or a vaginal plug. Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

Gestation day 5 to 19

Frequency of treatment:

daily

Duration of test:

20 days

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

21 – 24 mated females/dose in main study; 2 mated animals were additionally added to the high-dose and control groups for evaluation of zinc concentration in foetal tissue.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a dose range finding study conducted by the authors. In the dose-range finding study, 14-day repeated oral dose of ZnO(SM20(+)) NPs showed decreases in body weight, and changes in haematology and biochemistry parameters for 1000- and 2000-mg/kg/day groups. Death occurred in 2 male rats from the 2000-mg/kg/day group. Decreases in body weight, reductions in food consumption, and changes in haematology and biochemistry parameters were observed from 1000- and 2000-mg/kg/day groups. In a subchronic toxicity study of 90-day repeated oral treatment of ZnOSM20(+) NPs, the decreases in food and water consumptions were observed in 125-, 250-, and 500-mg/kg/day groups. Reductions in thymus weight, atrophy of testis and seminal vesicle, and changes of hematology and biochemistry parameters were present in 250- and 500-mg/kg/day groups. Moreover repeated oral doses of ZnOSM20(+) NPs caused aninar cell apoptosis, submucosal edema, inflammation in the glandular stomach, and decreases in total protein and albumin for 250- and 500-mg/kg/day groups. Either single oral dose or 90-day repeated oral doses (vehicle control and 125, 250, and 500 mg/kg/day) was performed for pharmacokinetic study; the increased zinc concentrations in plasma were seen for 125-, 250-, and 500-mg/kg/day groups (Chung, H. et al. (2013)). Therefore, for the prenatal and developmental toxicity study, the high dose was set to 400 mg/kg/day of body weight, and the middle and low doses was set to 200 and 100 mg/kg/day, respectively.

ZINC CONCENTRATION IN FOETAL TISSUE:
To investigate the placenta transfer of ZnO(SM20(+)) NPs in vivo, four extra female rats were used in the control (control group; n=2) and 400-mg/kg/day groups (ZnO(SM20(+)) NPs; n=2). Dosing occurred for the period of GD 5 – 19 in the same manner as that for the main study animals.

*Reference:
- Chung, H., Yu, J., Baek, M. et al. Toxicokinetics of zinc oxide nanoparticles in rats. J Phys Conf Ser. 2013; 429:012037.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily throughout the gestation period
- Cage side observations checked: mortality/morbidity and clinical signs (general appearance and behaviour)

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily from gestation day 0 to 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined on gestation days 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 and mean daily diet consumption calculated: Yes

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- All females were sacrificed by isoflurane inhalation and exsanguination for gross post mortem examination and caesarean section.
- Absolute and relative organ weights were determined for the following organs: liver, heart, brain, kidneys, ovaries, spleen, lung, gravid uterus, uteral cornua, adrenal glands, and pituitary gland.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Live/dead foetuses were counted.
- Histopathology: uteri with no evidence of implantation were stained with a 2% sodium hydroxide solution to identify the presence of early resorption sites. If no stained implantation site was present, the rat was considered “not pregnant”.

Blood sampling:

Not specified

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- The other half was preserved in Bouin solution and examined for internal softtissue changes using a freehand razor sectioning technique (Wilson, J.G. et al. (1965)) and Nishimura’s method (Suzuki, M. et al. (1973)).* The observed fetal morphological alterations in this study were classified as developmental malformations or variations. A malformation was defined as a permanent structural change that is likely to adversely affect survival or health.
- Skeletal examinations: Yes: half per litter
- Half of the live fetuses from each litter were fixed in absolute ethanol, eviscerated, and then processed for skeletal staining with alizarin red S and Alcyan blue using for subsequent skeletal examination (Menegola, E. et al. (2001)).*
- Anogenital distance of all live rodent pups: No data
- All live foetuses were weighed individually and sexed.

ZINC CONCENTRATION IN FOETAL TISSUE:
On GD 20, foetuses were collected via caesarean sections from dams, and Zn contents in the foetal tissues were analysed. Foetuses were digested in concentrated nitric acid overnight. The next day, nitric acid and perchloric acid were added to each sample and heated at 200 - 250 °C until the solutions were colourless and clear. The concentrated sample solutions were transferred into a 100-mL volumetric flask and filled with purified water to the measuring line. Before analysis, ICP-AES was calibrated every time by running at least six Zn standard concentrations (0.5, 2, 5, 10, 20, and 40 mg/L).

*References:
- Wilson JG. Methods for administering agents and detecting malformations in experimental animals. Teratology: Principles and Techniques. 1965: 262–277.
- Suzuki, M., Murakami, U., Tabuchi, A. Abstracts of papers presented at the thirteenth annual meeting of the congenital anomalies research association of Japan. Hiroshima, Japan, July 12–13, 1973. Teratology. 1973; 8(1): 83–111.
- Menegola, E., Broccia, M.L., Giavini, E. Atlas of rat fetal skeleton double stained for bone and cartilage. Teratology. 2001; 64(3): 125–133.

Statistics:

The unit for statistical measurement was the pregnant female or the litter (Weil, S.S. et al. (1970)).* Quantitative continuous data, such as the maternal body weight, food consumption, foetal body weight, and placental weight, were subjected to a one-way analysis of variance, and a Scheffe’s multiple comparison test was carried out for the significant differences (Scheffe, H. et al. (1953)).* The number of corpora lutea, total implantations, live and dead foetuses, and foetal alterations were evaluated statistically by using the Kruskal–Wallis nonparametric analysis of variance (Kruskal, W.H. et al. (1952)), followed by the Mann–Whitney U-test where appropriate.* The sex ratio and the proportions of litters with malformations and developmental variations were compared using a chi-square test and Fisher’s exact probability test (Fischer, R.A. (1970)).* Statistical analyses were performed by comparing the treatment groups with the control group using SPSS 19.0 software. Differences with a P-value ≤ 0.05 were considered to be statistically significant.

*References:
- Weil, C.S. Selection of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis or carcinogenesis. Food Cosmet Toxicol. 1970; 8(2): 177–182.
- Scheffe, H. A method for judging all contrasts in the analysis of variance*.
Biometrika. 1953; 40(1–2): 87–110.
- Kruskal WH, Wallis WA. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association. 1952; 47: 583–621.
- Fisher RA. Statistical methods for research workers. Oliver and Boyd; 1970.

Indices:

Preimplantation loss (%)= (number of corpora lutea - number of implantations) / (number of corpora lutea) x 100

Postimplantation loss (%)= (number of implantations - number of live foetuses) / (number of implantations) x 100

Foetal death = resorptions + dead foetuses

Historical control data:

Not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 100, 200 and 400 mg/kg bw/day: 7 of 24 dams from the low-dose group, 12 of 21 dams from the mid-dose group, and 18 of 23 dams from the high-dose group showed salivation around the mouth in general appearance.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Endocrine 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, treatment-related

Description (incidence and severity):

- 400 mg/kg bw/day: increased absolute and relative weights of adrenal gland were significant in a dose-dependent manner in comparison with the vehicle control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 400 mg/kg bw/day: hypertrophy of adrenal and lung, oedematous bowel, gastro-tympanites, and red reaction of liver were observed in dams (data not shown).

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS:
- 100, 200 and 400 mg/kg bw/day: starting from 3 - 12 days after oral administration, alopecia (localized areas of partial alopecia) was observed in one pregnant rat from the low-dose group, one from the mid-dose group, and one from the high-dose group (data not shown). This clinical sign was not recovered for the treatment period.

BODY WEIGHT AND WEIGHT CHANGES:
- 400 mg/kg bw/day: significant decreases in maternal body weight on GD 20 from the high-dose group was observed in comparison with the vehicle control group. The body weights of these pregnant females (406.7 ± 31.27 g) were reduced by 7 % compared to the controls (433.3 ± 23.90 g).The maternal-body-weight gain (121.2 ± 27.23 g) during pregnancy and corrected body weight (325.5 ± 26.35 g) were also significantly lower in the high-dose group than in the control group (148.4 ± 16.58 g and 349.0 ± 21.11 g; p<0.01).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 200 and 400 mg/kg bw/day: statistically significant decreases in food consumption were noticed on day 18 of gestation in the mid- and high-dose groups (26.21 ± 4.17 g and 25.87 ± 4.75 g) in comparison to the vehicle control group (31.55 ± 3.60 g; p<0.05).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- 400 mg/kg bw/day: significantly decreased absolute liver weight was observed, but no significant difference in the relative liver weight was observed compared to the control.

GROSS PATHOLOGICAL FINDINGS:
- 0 mg/kg bw/day: one case of caveola of kidney surface in the vehicle control group was observed in dams (data not shown).
- 200 mg/kg bw/day: one case of splenomegaly in the mid-dose group was observed in dams (data not shown).

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

TOTAL LITTER LOSSES BY RESORPTION
- totally resorbed litters were not found in any group.

EARLY OR LATE RESORPTION:
- 0, 100, 200 and 400 mg/kg bw/day: no significant changes in early or late resorptions were observed between all treatment and control groups.

DEAD FOETUSES:
- 0, 100, 200 and 400 mg/kg bw/day: the number of foetal deaths were similar for the treatment groups and the vehicle control group.

CHANGES IN NUMBER OF PREGNANT:
- Pregnancy rates were similar for all dosage groups, ranging from 87.5 – 100 %.

NUMBER OF IMPLANTATIONS AND IMPLANTATION RATES:
- 0, 100, 200 and 400 mg/kg bw/day: number of implantations and implantation rates were similar for all treatment groups and the vehicle control group.

FURTHER OBSERVATIONS:
- 0, 100, 200 and 400 mg/kg bw/day: number of corpora lutea and placental weight were similar for the treatment groups and the vehicle control group.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

gross pathology

organ weights and organ / body weight ratios

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Description (incidence and severity):

- 400 mg/kg bw/day: there were significant increases in the number of foetuses with visceral variations, such as misshapen thymus, ureter abnormality (grade III: very short or no papillae and a marked dilation of the renal space), and ectopic kidney.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

FOETAL BODY WEIGHT CHANGES:
- 400 mg/kg bw/day: significantly decreased foetal weights of males and females were observed in comparison to the vehicle control group. No dose dependency was observed.

CHANGES IN SEX RATIO:
- 0, 100, 200 and 400 mg/kg bw/day: the number of sex ratios of the live foetuses were similar for the treatment groups and the vehicle control group.

CHANGES IN LITTER SIZE AND WEIGHTS:
- 0, 100, 200 and 400 mg/kg bw/day: no significant changes in litter size were observed between treatment and control groups.

EXTERNAL MALFORMATIONS:
- 0, 100, 200 and 400 mg/kg bw/day: no foetus showed an external malformation.
- 0, 100, 200 and 400 mg/kg bw/day: haematoma was shown in all groups, including the vehicle control group. However, the numbers of foetuses with haematomas were not significantly increased in comparison to the control group.

SKELETAL MALFORMATION:
- 0, 100, 200 and 400 mg/kg bw/day: skeletal malformation, such as cleavage ossification of thoracic centrum, was observed in all groups. Although several types of skeletal variations were observed, including incomplete ossification of skull, dumbbell ossification of thoracic centrum, incomplete ossification of thoracic centrum, asymmetric thoracic centrum, supernumerary rib, short rib, and incomplete ossification of sternebrae, no significant difference in the number of foetuses with skeletal variations or in the number of affected foetuses was seen between the groups.

VISCERAL VARIATIONS:
- 0, 100, 200 and 400 mg/kg bw/day: several types of visceral variations were seen in foetuses of the treatment groups, including misshapen thymus, ureter abnormality dilated renal pelvis, and urinary bladder hypertrophy.

ZINC CONCENTRATION IN FOETAL TISSUE:
- 0 and 400 mg/kg bw/day: Zn contents in foetuses after in utero exposure to ZnO(SM20(+)) NPs was not significantly different from the Zn contents in control foetuses.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: visceral variations

Developmental effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Conclusions:

In this RDT study, positively charged ZnO nanoparticles, suspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer with 2 % L-serine as vehicle, were repeatedly administered by gavage to pregnant female SD rats (n=21 to 24/group) at doses of 100, 200 and 400 mg/kg bw/day over the period of gestation day 5 to 19.
Toxicity in the dams manifested as significantly decreased liver weight and increased adrenal glands weight after administration of 400 mg/kg/day NPs. However, no treatment-related difference in: number of corpora lutea; number of implantation sites; implantation rate (%); resorption; dead foetuses; litter size; foetal deaths and placental weights; and sex ratio were observed between the groups. On the other hand, morphological examinations of the foetuses demonstrated significant differences in incidences of abnormalities in the group administered 400 mg/kg/day. Meanwhile, no significant difference was found in the Zn content of foetal tissue between the control and high-dose groups.
Based on the authors, the significant toxic changes on intrauterine foetal growth and development were observed in the 400 mg/kg bw/day group and therefore a dosage level of 200 mg/kg/day of ZnO(SM20(+)) NPs was considered the NOAEL for both maternal toxicity and embryo–foetal development. Developmental effects are considered as secondary non-specific consequence of maternal toxicity effects.

The present study was conducted according to the OECD test guideline 414 (2001) and according to GLP. The methods and results are described appropriately, and the conclusions are plausible. However, the homogeneity of the test substance in the formulations which the animals received were not analysed. Gross pathological findings observed have not been tabulated by group. Individual data and historical control data were not presented. The pre- and postimplantation loss were determined, but the results were not shown. Gravid uterus was weighed, but it is unclear whether the cervix was included.
Therefore, the study is judged as reliable with restrictions because it represents a guideline study without detailed documentation.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Homogeneity of the test substance in the formulations which the animals received were not analysed. Clinical signs observed have not been tabulated by group or sex. No historical control data are available. The pre- and post-implantation loss were determined, but the results were not shown. Gravid uterus was weighed, but it is unclear whether the cervix was included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

yes

Remarks:

No analysis of homogeneity of test material in formulations; clinical signs observed have not been tabulated by group or sex; no historical control data; no results of pre- and postimplantation loss; unclear whether gravid uterus was weighed with cervix.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Sumitomo-Osaka Cement Co., Ltd., Tokyo, Japan; Lot no. 141319

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Inc., Gyeonggi-do, Korea
- Age at study initiation: 12 weeks old (10 weeks old at purchase + 12 days of quarantine and acclimation)
- Weight at gestation day 0 (mean values): 268.5 ± 13.16 g (control group); 271.5 ± 15.10 g (low-dose group); 269.7 ± 17.40 g (mid-dose group) and 266.2 ± 13.99 g (high-dose group)
- Housing: mated females were housed individually in clear polycarbonate cages with stainless steel wire lids.
- Diet (ad libitum): commercial rodent chow, supplier: Cargill Agri Purina, Inc., Gyeonggi-do, Korea
- Water (ad libitum): sterilized tap water after UV irradiation
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.8 - 23.0 °C
- Relative humidity: 45.3 - 56.9 %
- Air changes: 10 - 15 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:

oral: gavage

Vehicle:

other: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer

Remarks:

with 1 % sodium citrate

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
NPs were suspended in 20 nM HEPES buffer containing 1% sodium citrate (vehicle) and then mixed well. The final pH of the buffer solution was adjusted with 1 M sodium carbonate, and 4 % of the surface-modified ZnO(SM20(-)) NPs were used as a stock solution. Before administration, the suspension was stirred for 10 seconds and then diluted with distilled water.
- Dosing volume: 10 mL/kg (The daily application volume was calculated in advance, based on the most recently recorded body weight of the individual animal.)

VEHICLE
- Justification for use and choice of vehicle: to cap ZnO NPs with citrate molecules which are widely used capping agents for inorganic NPs for enhancing their surface charge property (Wang, J. et al. (2007), Wang, B. (2008) and Surekha, P. et al. (2012)).* Citrate was also used to reduce the aggregation and sedimentation of NPs and to obtain a well-dispersed suspension of ZnO NPs for oral administration (Leeuwenburgh, S.C. et al. (2010)).*

*References:
- Wang, J., Zhou, G., Chen, C., et al. Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administra¬tion. Toxicol. Lett. 2007; 168(2):176-185.
- Wang, B., Feng, W., Wang, M., et al. Acute toxicological impact of nano- and submicro-scaled zinc oxide powder on healthy adult mice. J Nanopart. Res. 2008; 10:263-276.
- Surekha, P., Kishore, A.S., Srinivas, A., et al. Repeated dose dermal toxicity study of nano zinc oxide with Sprague-Dawley rats. Cutan. Ocul. Toxicol. 2012; 31(1): 26-32.
- Leeuwenburgh, S.C., Ana, I.D., Jansen, J.A. Sodium citrate as an effective dispersant for the synthesis of inorganic-organic composites with a nanodispersed mineral phase. Acta Biomater. 2010; 6(3): 836-844.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the dosing solution was measured on gestational days (GDs) 5, 11, and 19 using ICP-AES.
Results:
Concentrations: three analyses confirmed that the concentrations of all dose formulations were within ±15 % of the target concentrations. Concentrations of total Zn were 9.83 ± 0.47 mg/mL for the 100 mg/kg/day group, 19.35 ± 1.86 mg/mL for the 200 mg/kg/day group, and 39.86 ± 1.51 mg/mL for the 400 mg/kg/day group.
Stability: ZnO(SM20(-)) NPs were stable for 4 hours at room temperature.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male / 1 female
- Proof of pregnancy: each morning, the female rats were examined for the presence of sperms or a vaginal plug. Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

gestation day 5 to 19

Frequency of treatment:

daily

Duration of test:

20 days

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

21 – 25 mated females/dose in main study; 2 mated females were additionally added to the high-dose and control groups for evaluation of zinc concentration in foetal tissue.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a dose range finding study conducted by the authors. In the previous dose range finding study, a 14-day repeated oral dose of ZnO(SM20(-)) NPs showed a decrease of body weight and changes in haematology and biochemistry parameters for 1000 mg/kg/day and 2000 mg/kg/day groups. In the case of ZnO(SM20(+)), death was observed in two male rats from the 2000 mg/kg/day group. Body weight decrease, food consumption loss, and change of haematology and biochemistry parameters were observed from the 1000 mg/kg/day and 2000 mg/kg/day groups. In a subchronic toxicity study of 90 days of repeated oral treatment of ZnOSM20(-), decreased food and water consumption was observed from the 125 mg/kg/day, 250 mg/kg/day, and 500 mg/kg/day groups, and weight reduction of the thymus, atrophy of the testis and seminal vesicle, as well as changes in the hematology and biochemistry parameters were observed from the 250 mg/kg/day and 500 mg/kg/day groups. Moreover, a repeated oral dose of ZnOSM20(+) caused aninar cell apoptosis, submucosal edema, and inflammation in the glandular stomach, as well as decreases in the total protein and albumin for the 250 mg/kg/day and 500 mg/kg/day groups. When either a single oral dose or 90-day repeated oral dose (vehicle control, 125 mg/kg/day, 250 mg/kg/day, and 500 mg/kg/day) study was performed for pharmacokinetic investigations, Zn concentrations in plasma increased among the 125 mg/kg/day, 250 mg/kg/day, and 500 mg/kg/day groups (Chung, H.E. et al. (2013)).* Therefore, for the prenatal and developmental toxicity study, the high dose was set to 400 mg/kg/day of body weight and the middle and low doses were set to 200 mg/kg/day and 100 mg/kg/day, respectively.

ZINC CONCENTRATION IN FOETAL TISSUE STUDY:
To investigate the placenta transfer of ZnO(SM20(-)) NPs in vivo, four extra female rats were used in the control (n=2) and 400 mg/kg/day groups (ZnO(SM20(-)) NPs; n=2), respectively. Dosing occurred for the period of GDs 5 – 19 in the same manner as was used for the main study animals.

*Reference:
- Chung, H.E., Yu, J., Baek, M. et al. Toxicokinetics of zinc oxide nanoparticles in rats. J Phys Conf Ser. 2013: 429.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily throughout the gestation period
- Cage side observations checked: mortality/morbidity and clinical signs (general appearance and behaviour).

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily from gestation day 0 to 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined on gestation days 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 and mean daily diet consumption calculated as g food/day: Yes

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- All females were sacrificed by isoflurane inhalation and exsanguination for gross post mortem examination and caesarean section.
- Absolute and relative organ weights were determined for the following organs: liver, heart, brain, kidneys, ovaries, spleen, lung, gravid uterus, uteral cornua, adrenal glands, and pituitary gland.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Live/dead foetuses were counted.
- The uteri with no evidence of implantation were stained with a 2 % sodium hydroxide solution to identify the presence of early resorption sites. If no stained implantation sites were present, the rat was considered “not pregnant”.

Blood sampling:

- Plasma: No data
- Serum: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- The other half was preserved in Bouin’s solution and examined for internal soft tissue changes, using a freehand razor sectioning technique (Wilson, J.G. et al. (1965)) and Nishimura’s method Nishimura, K.A. et al. (1974)).*
- Skeletal examinations: Yes: half per litter
- Half of the live fetuses from each litter were fixed in absolute ethanol, eviscerated, and then processed for skeletal staining with Alizarin red S and Alcyan blue, and were used for subsequent skeletal examination (Menegola, E. et al. (2001)).*
- Anogenital distance of all live rodent pups: No data
- All live fetuses were weighed individually, and their sexes were determined.

ZINC CONCENTRATION IN FOETAL TISSUE STUDY
On GD 20, foetuses were collected by caesarean sections from dams, and Zn contents in the foetal tissues were analysed. Foetuses were digested in concentrated nitric acid overnight. The next day, nitric acid and perchloric acid were added to each sample and heated at 200°C - 250°C until the solutions were colourless and clear. The concentrated sample solutions were put into a 100 mL volumetric flask, which was filled with purified water to the marked line. Before analysis, ICP-AES was calibrated every time by running at least six Zn standard concentrations (0.5, 2, 5, 10, 20 and 40 mg/L).

*References:
- Wilson, J.G. Methods for administering agents and detecting malformations in experimental animals. In: Wilson JG, Warkany J, editors. Teratology, Principles and Techniques. Chicago, IL: University of Chicago Press; 1965: 262–277.
- Nishimura, K. A microdissection method for detecting thoracic visceral malformations in mouse and rat fetuses. Congenit Anom (Kyoto). 1974; 14:23–40.
- Menegola, E., Broccia, M.L., Giavini, E.. Atlas of rat fetal skeleton double stained for bone and cartilage. Teratology. 2001; 64(3): 125–133.

Statistics:

The evaluation item, the statistical analysis was performed on the pregnant rats or foetuses (Weil, C.S. et al. (1970)).* Quantitative continuous data, such as maternal body weight, food consumption, foetal body weight, and placental weight, were subjected to a one-way analysis of variance (ANOVA), and a Scheffe’s multiple comparison test was carried out when the differences were significant (Scheffe, H. et al. (1953)).* The number of corpora lutea, total implantations, live and dead foetuses, and foetal alterations were statistically evaluated using the Kruskal–Wallis nonparametric ANOVA (Kruskal, W.H. et al. (1952)), followed by the Mann–Whitney U-test, where appropriate.* The sex ratio and the proportions of litters with malformations and developmental variations were compared using a chi-square test and Fisher’s exact probability test (Fischer, R.A. et al. (1970)).* Statistical analyses were performed by comparing the treatment groups with the control group using SPSS 19.0 software. A difference with a P-level ≤ 0.05 was considered statistically significant.

*References:
- Weil, C.S. Selection of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis or carcinogenesis. Food Cosmet Toxicol. 1970; 8(2): 177–182.
- Scheffé, H. A method for judging all contrasts in the analysis of variance*. Biometrika. 1953; 40(1/2): 87–110.
- Kruskal, W.H, Wallis, W.A. Use of ranks in one-criterion variance analysis. J Am Stat Assoc. 1952; 47(260): 583–621.
- Fisher RA. Statistical Methods for Research Workers. 14th ed. Edinburgh, Scotland: Oliver and Boyd; 1970.

Indices:

Preimplantation loss (%):
[n of corpora lutea – n of implantations/n of corpora lutea] × 100

Postimplantation loss (%):
[n of implantations – n of live foetuses/n of implantations] × 100

Foetal death = Resorptions + Dead foetuses.

Historical control data:

Not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Six of 21 dams from the 100 mg/kg/day group, ten of 23 dams from the 200 mg/kg/day group, and 16 of 25 dams from the 400 mg/kg/day group showed salivation around mouth in terms of their general appearance, but it was transiently observed after treatment.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Endocrine 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, treatment-related

Description (incidence and severity):

- 200 and 400 mg/kg bw/day: the relative liver weight in the mid-dose group, and the absolute and relative liver weights in the high-dose group were significantly decreased in a dose-dependent manner in comparison with those of the vehicle control group. Absolute and relative weights of the right adrenal gland the mid- and high-dose groups and relative weights of the left adrenal gland in the high-dose group were significantly increased in a dose-dependent manner in comparison with the vehicle control group.

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS:
- 0, 100, 200 and 400 mg/kg bw/day: alopecia (localized areas of partial alopecia) was observed in 2 pregnant rats from the vehicle control group, 1 from the 100 mg/kg/day group, 1 from the 200 mg/kg/day group, and 2 from the 400 mg/kg/ day group, starting from 6 – 10 days after oral administration (data not shown). This clinical sign was not recovered during the treatment period.

MORTALITY:
- no death of maternal animals occurred.

BODY WEIGHT AND WEIGHT CHANGES:
- 400 mg/kg bw/day: the maternal body weight on GD 20 in the high-dose group was significantly decreased when compared with the vehicle control group. The body weight of these pregnant females (406.4 ± 23.97 g) was reduced by 4 % compared to controls (422.2 ± 20.86 g; p<0.05). The maternal body weight gain during pregnancy (140.2 ± 20.11 g; p<0.05) and corrected body weight (320.6 ± 21.14 g; p<0.01) were also significantly lower than those for the control group (153.8 ±13.93 g and 340.5 ± 19.77 g).

FOOD CONSUMPTION:
- there was no statistically significant difference in food consumption between the control and treatment groups.

GROSS PATHOLOGICAL FINDINGS:
- no treatment-related gross finding was observed in the dams from all groups at the scheduled autopsy.

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

TOTAL LITTER LOSSES BY RESORPTION
- 0, 100, 200 and 400 mg/kg bw/day: no totally resorbed litter was found in any group.

EARLY OR LATE RESORPTION:
- 0, 100, 200, and 400 mg/kg bw/day: there was no test-item related change in early or late resorptions.

DEAD FOETUSES:
- 0, 100, 200 and 400 mg/kg bw/day: the number of foetal deaths were similar for the treatment groups and the vehicle control group.

CHANGES IN NUMBER OF PREGNANT
- the overall pregnancy rates were similar for all dosage groups, ranging from 84 - 100 %. There were 22 pregnant females in the control group, 21 in the low-dose group, 24 and 25 in the mid-dose and high-dose group.

IMPLANTATION
- 0, 100, 200, and 400 mg/kg bw/day: the number of implantations and implantation rates were similar for the treatment groups and the vehicle control group.

FURTHER OBSERVATIONS:
- 0, 100, 200, and 400 mg/kg bw/day: no significant difference between the treatment and control groups was seen for placental weight.
- 0, 100, 200, and 400 mg/kg bw/day: there was no test-item related change in corpora lutea.

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

FOETAL BODY WEIGHT CHANGES:
- 0, 100, 200 and 400 mg/kg bw/day: no significant difference between the treatment and control groups was seen for foetal weight.

CHANGES IN LITTER SIZE AND WEIGHTS:
- 0, 100, 200 and 400 mg/kg bw/day: no significant difference between the treatment and control groups was observed for litter size.

EXTERNAL MALFORMATIONS:
- 0, 100, 200 and 400 mg/kg bw/day: there were no foetuses with external malformations.
- 0, 100, 200 and 400 mg/kg bw/day: the observed external variations in the treatment groups were haematoma (treatment group: n=2-4; control group: n=6/22), hyperplasia of the tail only in low-dose group (n=1/21), and short tail in high-dose group (n=1/25). However, the numbers of foetuses with external variations were not significantly increased in comparison to the control group.

SKELETAL MALFORMATION:
- 100, 200 and 400 mg/kg bw/day: there were some types of skeletal variations, including incomplete ossification of skull, dumbbell ossification of the thoracic centrum, bipartite ossification of the thoracic centrum, asymmetric thoracic centrum, supernumerary rib, asymmetric sternebrae, incomplete ossification of the sternebrae, and incomplete lumbar ossification, no significant difference was observed in the number of foetuses with skeletal variations or in the number of litters with affected foetuses between the groups.

VISCERAL MALFORMATIONS:
- 100, 200 and 400 mg/kg bw/day: there were some types of visceral variations, including misshapen thymus, ureter abnormality, dilated renal pelvis, large kidney, and ectopic kidney in the foetuses of the treatment groups, no significant difference was found in the number of foetuses with visceral variations or in the number of litters with affected foetuses between the groups.

ZINC CONCENTRATION IN FOETAL TISSUE:
- 0 and 400 mg/kg bw/day: the measured total Zn levels was 14.44 ± 0.37 μg/g for the control group, and 19.02 ± 0.60 μg/g (ZnO(SM20(-)) NPs) for the 400 mg/kg/day group. The Zn contents in foetuses after in utero exposure to ZnO(SM20(-)) NPs were not significantly different from the Zn contents in control foetuses.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

no

Conclusions:

In this RDT study, negatively charged ZnO nanoparticles, suspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer with 1 % sodium citrate, were repeatedly administered by gavage in pregnant female SD rats (n=21 to 25/group) at doses of 100, 200 and 400 mg/kg bw/day over the period of gestation day 5 to 19.
Toxicity in the dams manifested as significantly decreased liver weight, and increased adrenal glands weight at 200 mg/kg/day and 400 mg/kg/day. However, no treatment-related difference in the number of corpora lutea, the number of implantation sites, the implantation rate (%), resorption, dead foetuses, litter size, foetal deaths, foetal and placental weights, and sex ratio were observed between the groups. Morphological examinations of the foetuses demonstrated no significant difference in the incidences of abnormalities between the groups. No significant difference was found in the Zn content of foetal tissue between the control and high-dose groups.
Based on the authors, a dosage level of 100 mg/kg bw/day of negatively charged ZnO NPs was considered the NOAEL for maternal toxicity and the highest dose level of 400 mg/kg bw/day was considered the NOAEL for embryo–foetal development.

The present study was conducted according to the OECD test guideline 414 (2001) and according to GLP. The methods and results are described appropriately, and the conclusions are plausible. However, the homogeneity of the test substance in the formulations which the animals received were not analysed. Individual data and historical control data were not presented. The pre- and post-implantation loss were determined, but the results were not shown. Gravid uterus was weighed, but it is unclear whether the cervix was included.
Therefore, the study is judged as reliable with restrictions because it represents a guideline study without detailed documentation.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

chronic

Species:

other: rat, mouse, rabbit, hamster

Quality of whole database:

guideline studies available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

Study duration:

chronic

Species:

rat

Quality of whole database:

guideline study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Animal studies – developmental toxicity

Oral
In this developmental toxicity study (Hong et al 2014a) in rats conducted according to OECD guideline 414 and under GLP, the effects of negatively charged ZnO NP on dams and foetuses were investigated after oral administration (gavage) from gestation day 5 to 19. Test material: ZnO NPs negatively charged (ZnOSM20(-) NPs) (Sumitomo-Osaka Cement Co., Ltd. (Tokyo, Japan) capped with citrate molecules (Lot. No. 141319, Particle size: 20 nm)
For oral administration of ZnO NPs were suspended in 20 nM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer containing 1% sodium citrate (vehicle). Pregnant Crl:CD (SD) rats (12 weeks of age, Orient Bio Inc., Gyeonggi-do, Korea) were subjected to three treatment groups which received ZnO NPs at 100 (n=21), 200 (n=24) and 400 mg/kg bw/day (n=25) and one vehicle control group (n=22). The test mixture was administered daily by gavage to the pregnant rats from GD 5 through GD 19 with a dose volume of 10 mL/kg body weight. The vehicle control group received only a HEPES/citrate buffer solution with gavage. On GD 20, dams were subjected to caesarean section. This study was performed in compliance with OECD) test guideline 414 (2001) and in accordance with the Good Laboratory Practice (GLP) principles. General toxicity tests were performed in dams and development-related endpoints were investigated in foetuses. Tissue samples were analysed for Zn concentrations.
Three analyses confirmed that the concentrations of all dose formulations were within ±15% of the target concentrations. ZnO NPs were stable for 4 hours at room temperature. The measured total Zn levels was 14.44±0.37 μg/g (mean ± standard deviation) for the control group, and 19.02±0.60 μg/g for the 400 mg/kg/day group. The Zn contents in foetuses after in utero exposure to Zn NPs were not significantly different from the Zn contents in control foetuses.
Salivation was observed in all treated groups, but not considered to be related to the ZnO NP treatment, since salivation was observed sporadically and was not dose dependent. The maternal body weight on GD 20 in the high-dose group was significantly decreased when compared with the vehicle control group. The maternal body weight gain during pregnancy (P<0.05) and corrected body weight (P<0.01) were also significantly lower than those for the control group. There was no statistically significant difference in food consumption between the control and treatment groups. At the scheduled autopsy, no treatment-related gross finding was observed in the dams from all groups. The relative liver weight in the 200 mg/kg/day group, and the absolute and relative liver weights in the 400 mg/kg/day group were significantly decreased in a dose-dependent manner in comparison with those of the vehicle control group. Absolute and relative weights of the right adrenal gland in the 200 mg/kg/day and 400 mg/kg/day groups and relative weights of the left adrenal gland in the 400 mg/kg/day group were significantly increased in a dose-dependent manner. The overall pregnancy rates were similar for all dosage groups, ranging from 84%–100%. No totally resorbed litter was found in any group. The number of corpora lutea, implantations, implantation rates, foetal deaths, and sex ratios of the live foetuses were similar for the treatment groups and the vehicle control group. No significant difference between the treatment and control groups was seen for placental weight and foetal weight. There were no foetuses with external, visceral or skeletal malformations. A few external, visceral and skeletal variations were observed but regarded as not related to treatment with ZnO NPs.
The results of this developmental toxicity study suggested that the administration of negatively charged ZnO NPs (ZnOSM20(-) NPs) to pregnant rats had no impact on embryo–foetal development in rats. Maternal toxicity was evident based on effects on body weights and organ weights at 200 and 400 mg/kg bw/d. Based on the results of this study, a dosage level of 100 mg/kg bw/day of negatively charged ZnO NPs was considered the no-observed-adverse-effect level for maternal toxicity. The highest dose level of 400 mg/kg bw/day was considered the no-observed-adverse-effect level for embryo–foetal development.
The results of this developmental toxicity study in SD rats can generally be regarded as reliable with restrictions, because the study was conducted based on the OECD guideline 414 (2001) and according to GLP. The methods and results are described appropriately, and the conclusions are plausible. Therefore, the study is judged as reliable with restrictions [RL=2] because it is represents a guideline study without detailed documentation.

In this developmental toxicity study (Hong et al 2014b) in rats conducted according to OECD guideline 414 and under GLP, the effects of positively charged ZnO NP on dams and foetuses were investigated after oral administration (gavage) from gestation day 5 to 19. Test material: ZnO NPs positively charged (ZnOSM20(+) NPs) (Sumitomo-Osaka Cement Co., Ltd. (Tokyo, Japan) capped with L-serine molecules (Lot: No 141319, Particle size: 20 nm).
For oral administration of ZnO NPs were suspended in 20 nM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer containing 1% sodium citrate (vehicle). Pregnant Crl:CD (SD) rats (12 weeks of age, Orient Bio Inc., Gyeonggi-do, Korea) were subjected to three treatment groups which received ZnO NPs at 100 (n=24), 200 (n=21) and 400 mg/kg bw/day (n=23) and one vehicle control group (n=23). The test mixture was administered daily by gavage to the pregnant rats from GD 5 through GD 19 with a dose volume of 10 mL/kg body weight. The vehicle control group received only a HEPES/citrate buffer solution with gavage. On GD 20, dams were subjected to caesarean section.
This study was performed in compliance with OECD) test guideline 414 (2001) and in accordance with the Good Laboratory Practice (GLP) principles. General toxicity tests were performed in dams and development-related endpoints were investigated in foetuses. Tissue samples were analysed for Zn concentrations.
Three analyses confirmed that the concentrations of all dose formulations were within ±15% of the target concentrations. ZnO NPs were stable for 4 hours at room temperature. The measured total Zn levels was 14.44±0.37 μg/g (mean ± standard deviation) for the control group, and 16.47±2.19 μg/g for the 400 mg/kg/day group. The Zn contents in foetuses after in utero exposure to Zn NPs were not significantly different from the Zn contents in control foetuses.
Salivation was observed in all treated groups, but not considered to be related to the ZnO NP treatment, since salivation was observed sporadically and was not dose dependent. Significant decreases in maternal body weight on GD 20 from the high-dose group was observed in comparison with the vehicle control group. The maternal-body-weight gain during pregnancy and corrected body weight were also significantly lower in the high-dose group than in the control group. Statistically significant decreases in food consumption were noticed on day 18 of gestation in the 200 and 400 mg/kg bw/day groups in comparison to the vehicle control group. At the scheduled autopsy, one case of caveola of kidney surface in the vehicle control group; one case of splenomegaly in the 200 mg/kg bw/day group; and hypertrophy of adrenal and lung, oedematous bowel, gastro-tympanites, and red reaction of liver in the 400 mg/kg bw/day group were observed in dams. Significantly decreased absolute liver weight was observed in the 400 mg/kg bw/day group was observed, and the increased absolute and relative weights of adrenal gland were significant in the 400 mg/kg/day group in a dose-dependent manner in comparison with the vehicle control group. The overall pregnancy rates were similar for all dosage groups, ranging from 87.5%–100%. Totally resorbed litters were not found in any group. The number of corpora lutea, implantations, and foetal deaths, as well as implantation rates, placental weight, and sex ratios of the live foetuses were similar for the treatment groups and the vehicle control group. Significantly decreased foetal weights of males and females were observed in the 400 mg/kg bw/day group in comparison to the vehicle control group. No foetus showed an external or visceral malformation. Several types of visceral variations were seen in foetuses of the treatment groups. There were significant increases in the number of foetuses with visceral variations, such as misshapen thymus, ureter abnormality (grade III), and ectopic kidney in the 400 mg/kg bw/day group. Skeletal malformation, such as cleavage ossification of thoracic centrum, was observed in all groups. Although several types of skeletal variations were observed, no significant difference in the number of foetuses with skeletal variations or in the number of affected foetuses was seen between the groups.
The results of this developmental toxicity study suggested minimal effects of ZnOSM20(+) NPs on intrauterine growth and on foetal visceral morphology even at 400 mg/kg bw/d. Maternal toxicity was evident by effects on body weights, food consumption, organ weights and pathological changes at 400 mg/kg bw/d.
Based on the results of this study, a dosage level of 200 mg/kg/day of positively charged ZnOSM20(+) NP was considered the no-observed-adverse-effect level for both maternal toxicity and embryo–foetal development. Developmental effects are considered as secondary non-specific consequence of maternal toxicity effects.

The results of this developmental toxicity study in SD rats can generally be regarded as reliable with restrictions, because the study was conducted based on the OECD guideline 414 (2001) and according to GLP. The methods and results are described appropriately, and the conclusions are plausible. Therefore, the study is judged as reliable with restrictions [RL=2] because it represents a guideline study without detailed documentation.

In a test series, the developmental toxicity of zinc sulfate was assessed in hamster, mice, rabbits and rats (Food and Drug research, 1973):
Female hamsters (23-25 animals/group; outbred strain of golden hamster) received daily doses of 0.9, 4.1, 19 and 88 mg unspecified ZnSO4/kg bw by gavage during days 6-10 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 8, 10 and 14 of gestation. The females were sacrificed at day 14. The urogenital tract of each animal was examined in detail. Between 21 and 24 females were pregnant at term in all groups. No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups. Under the conditions of the test, administration of up to 88 mg/kg bw of unspecified zinc sulphate (ca. 35.2 mg or 19.9 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult hamsters and their foetuses.
Female CD-1 mice (25-30 animals/group) received daily doses of 0.3, 1.4, 6.5 and 30 mg unspecified ZnSO4/kg bw by gavage during days 6-15 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 6, 11, 15 and 17 of gestation. The females were sacrificed at day 17. The urogenital tract of each animal was examined in detail. No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups. Under the conditions of the test, administration of up to 30 mg/kg bw of unspecified zinc sulphate (ca.12 mg or 6.8 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult mice and their foetuses.
Female Dutch rabbits (14-19 animals/group) received daily doses of 0.6, 2.8, 13 and 60 mg unspecified ZnSO4/kg bw by gavage during days 6-18 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 6, 12, 18 and 29 of gestation. The urogenital tract of each animal was examined in detail. The females were sacrificed at day 29. Between 10 and 12 females were pregnant at term in all groups. No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups. Under the conditions of the test, administration of up to 60 mg/kg bw of unspecified zinc sulphate (ca. 24 mg or 13.6 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult rabbits and their foetuses.
Female Wistar rats (25-28 animals/group) received daily doses 0.4, 2.0, 9.1 and 42.5 mg unspecified ZnSO4/kg bw by gavage during days 6-15 of gestation. A control group was included. All animals were observed daily for appearance and behaviour with particular attention to food consumption and body weight. Body weights were recorded on day 0, 6, 11, 15 and 20 of gestation. The females were sacrificed at day 20. The urogenital tract of each animal was examined in detail. At term 25 females were pregnant in all groups. No clearly discernible effects on maternal survival, body weight gains, number of corpora lutea, implantations and resorptions were observed. The number of live litters, live and dead foetuses, the foetus weights and sex ratio were not affected by treatment. No difference in number or kind of abnormalities (in either soft or skeletal tissues) was found between exposed and control groups. Under the conditions of the test, administration of up to 42.5 mg/kg bw of zinc sulphate (ca. 17 mg or 9.6 mg Zn2+/kg bw, for anhydrate and heptahydrate, respectively) had no adverse effects on adult rats and their foetuses.
It is concluded that in the studies of prenatal developmental toxicity with zinc sulfate, no indications of maternal or developmental toxicity were found in mice, rats, hamsters and rabbits. Maximum zinc doses corresponding to 6.8 mg/kg body weight and day were used in mice, 200 mg/kg body weight and day in rats, 20 mg/kg body weight and day in hamsters, and 13.6 mg/kg body weight and day in rabbits.

In this reproductive and development toxicity screening study (non-GLP) in rats conducted according to OECD guideline 421, the effects of oral (gavage) exposure of male (2 weeks before mating) and female rats (2 weeks before mating to postnatal day 4) to one dose of zinc oxide nanomaterials was evaluated (Jo et al. 2013). Test material: ZnO NP (Sigma-Aldrich Korea Ltd., Yongin, Korea), Particle size: <100 nm (35 nm average particle size 50 wt % water). Specific-pathogen free male and female (7-weeks-old) SD rats (OrientBio Sungnam, Korea) were randomly divided into two groups and each group consisted of 12 male and 12 female rats. The test materials was administered by gavage at dose levels of 0 or 500 mg/kg bw/day to male rats for 6 weeks including 2 weeks before mating, and to females from 2 weeks before mating to day 4 of lactation including the gestation period. The dose was selected based on the results of the repeated dose 13-week oral toxicity study of zinc oxide nanoparticle. Throughout the test period, clinical observations were conducted once a day. Males and females were weighed on the first day of dosing, at least weekly thereafter, and at termination. The number of stillbirths, live births, runts and the presence of gross abnormalities of each litter were examined. On day 3 of lactation, live pups were counted and weighed. At the time of sacrifice, adult animals were examined macroscopically for any abnormalities or pathological changes. The testes, epididymis, ovaries and uterus of adult animals were weighed. A small piece of liver, kidney, uterus and mammary issue in dams and brain, liver, kidney, stomach including weaned milk and blood were collected and the zinc burdens in tissues were quantified. A small piece of testis and epididymis of male rats and ovary and uterus of female rats was processed for histopathology.
The rats administered with 500 mg/kg bw/d ZnO NP showed symptoms of diarrhoea and alopecia during the first 2 weeks of treatment. Three male rats in zinc oxide nanoparticles-group were found dead at 4, 13 and 25 days after administration. All mortalities were preceded by body weight loss and anorexia. The body weight gain of the treated rats by zinc oxide nanoparticles was about up to 16% lower than the control rats. There was no significant difference in the body weights and, no obvious difference was observed in the testes, epididymis and ovary weights between two groups. However, a significant (p<0.05) increase in the uterus weight was found in female rats treated 500 mg/kg bw/d. No effect on male and female fertility and mating/gestation period was observed. The number of implants was similar between groups, indicating that pregnancy rate, mating performance and post-implantation were unaffected. The number of newborn and live pups after day 4 of birth was reduced in the treated group. The number of pups surviving from birth to weaning was reduced gradually during lactation and pups gained less weight in the exposed group. At necropsy, offspring born to dams in treatment group had no abnormal incidence. A significant (p<0.05) increase in the zinc content was found in the mammary tissue of adult rats in the zinc oxide nanomaterials-treated group compared with control. The liver, kidney and uterus of female adult rats also showed increases in the zinc content but did not reach statistically significant due to high standard deviations. The offspring exposed to ZnO NPs showed significantly (p<0.05) higher levels of zinc contents in liver and kidney compared to control group. The zinc content of stomach including milk and blood in pups exposed to ZnO NPs increased, but it was not significantly different. In contrast, there was no difference in zinc content in the brain of treated and control pups. No abnormal findings were observed in the testes, epididymis and ovaries. In uterus, multi-focal granulation tissues were noted in both groups and considered to be the implantation site of foetus, and they were on the process of repairing after foetal delivery.
In this reproductive and development toxicity screening study in rats treated with ZnO NPs no effects on fertility and pregnancy outcome were observed, but the results showed effects on the offspring by a reduced number of born/live pups, decreased body weights of pups and increased foetal resorption.
Although this reproductive and development toxicity screening study was conducted in consideration of OECD guideline 421 it is only of supportive nature, because only one high dose level was chosen for evaluation and thus no dose-response or no effect level was determined. In addition, it needs to be taken into consideration that only very few signs of general toxicity were seen in female animals administered 500 mg/kg bw/d, although it is known from other studies, that such a high dose results in severe toxicity. However, the study appears to be well conducted and meets generally accepted scientific principles and the results are reported adequately, but it is not sufficient for an assessment because of the single high dose level chosen and can thus be regarded only as not reliable [RL=3].

The supporting study by Pal and Pal 1987 was conducted to determine the effect of post-coitum, and pre- and post-coitum dietary zinc supplementation on the conception in the Charles-Foster rat. In the post-coitum study (test 1), two groups of 15 pregnant rats were fed 0 and 4,000 ppm zinc as zinc sulphate in diet from day 1 through day 18 of pregnancy. In the pre- and post-coitum study (test 2), two groups of 15 female rats were treated with same doses for 21 day pre-mating period, maximum 5 day of mating period and 18 day of post-coitum period. All the females were sacrificed on day 18 of gestation and uterus content and foetuses were examined. In test 1, significant decrease in the incidences of conception and number of implantation sites per mated female was observed in the treatment group with respect to the control group. However, the difference in implantation sites when considered per pregnant female was not significant. In test 2, no significant difference in incidences of conception and implantation sites was observed in the control and treatment groups. In both the tests, there was no treatment-related change in the foetal and placental weights, stillbirths and malformed foetuses were absent and the number of resorption sites was negligible. Based on these results, dietary zinc supplementation at 4,000 ppm did not affect the foetal growth in pregnant rats. This dose, however, altered the normal conception when started after coitus but showed no effect when initiated sufficient time before coitus. The study is considered not reliable (RL=3) due to major deviations from the guideline for developmental toxicity testing, thus of supporting nature only.

Feng et al. (2017) examined the neurotoxicity of prenatal exposure to zinc oxide nanoparticles (diameter: approx. 50 nm) in rats. A group of ten pregnant Sprague Dawley rats received zinc oxide nanoparticles in saline containing 0.05 % v/v Tween 80 via oral gavage at a dose level of 500 mg/kg bw/day. The test item was administered daily for 18 consecutive days (gestation days 2 to 19). A vehicle control group was run concurrently (n = 10 pregnant rats). The dams were allowed to deliver their pups and the pups were investigated.
The results showed that the zinc content in the blood of the maternal animals was comparable to the zinc content of the control animals. Furthermore, body weight and relative organ (to bw) data obtained in 2-day-old pups showed that compared with the control group, the rats in the test item-treated group had lower body weights (p<0.01). Additionally, relative brain, heart and liver weights in the test item-treated group were significantly higher than those in the control group (p<0.05 or p<0.01). In contrast, relative kidney and spleen weights were remarkably decreased (p<0.01). Histopathological evaluation of the brains in 2-day-old offspring revealed that compared with the control offspring, the brain slices of rats prenatally treated with zinc oxide nanoparticles exhibited slight abnormalities with more sparse tissue in both brain regions (prefrontal cortex and hippocampus) observed in limited areas. Following administration of nanoparticles, the number of Ki-67 positively stained (brown) cells in the prefrontal cortex and hippocampus sharply decreased. Moreover, the nuclei of brain cells positively stained by TUNEL and 8-OhdG were significantly increased in both areas compared to that of the control pups. In addition, the ultrastructure of the neurons from zinc oxide nanoparticles-exposed rats (postnatal day 2) presented irregularity of the cell membrane, obvious mitochondrial swelling and autophagosomes. Evidence of cellular localization of nanoparticles was found in the neural synapse. For the 2-day-old pups, zinc significantly accumulated in the heart (p<0.01), liver (p<0.01), kidneys (p<0.01) and brain (p<0.05). The maximum amount of zinc was distributed in the liver. No obvious changes in the zinc concentration were observed in the lung and spleen between the control group and the treatment group. At weaning (postnatal day 21), the zinc contents in total blood were similar in the control and the offspring exposed to nanoparticles. Compared with the control group, the concentrations of reactive oxygen species (p<0.01) and malondialdehyde (p<0.05) were significantly increased in offspring exposed to zinc oxide nanoparticles at postnatal day 2. Furthermore, obvious decreases in superoxide dismutase (p<0.05) and glutathione peroxidase (p<0.01) activities were also observed in the brains from the nanoparticle-treated group. Prenatal exposure to zinc oxide nano-particles caused subtle but significant changes in genes in brains of newborns (postnatal day 2) and weaned offspring (postnatal day 21) as was observed in a total RNA extraction and real-time PCR. Lastly, the results of the behavioural testing using the Morris water maze conducted on postnatal day 60 showed that for all animals the latencies to reach the platform decreased over the course of the acquisition phase. Furthermore, on training days 1–3, there was no significant difference (p > 0.05) in escape latency to find the platform between the treatment group and the control group. However, exposure to zinc oxide nanoparticles exerted different effects in female and male offspring. Treatment with nanoparticles increased the latency of female offspring to reach the platform in fourth (p < 0.05) and fifth (p < 0.01) training days. During the first day of reacquisition training, female rats in the nanoparticle group also presented longer latency to reach the platform compared with the control rats. Moreover, the nanoparticle female offspring spent less time (p < 0.05) in the North-east quadrant (the former platform location) during the probe test, although the crossings over the former platform location remained unchanged (p > 0.05). On the other hand, treatment with zinc oxide nanoparticles did not alter the behavioural performance of nano-zinc oxide male offspring in the Morris water maze. In conclusion, the adverse effects on offspring brain, as described above, may cause impaired learning and memory capabilities in adulthood, particular in females.

Although the study performance and reporting of results appears to be adequate, the study shows significant methodological and reporting deficiencies. Firstly, the test substance was insufficiently described (purity and stability missing). Furthermore, the number of maternal animals (n = 10) chosen at the beginning was quite low compared to the recommend number of animals (n =20) by the OECD guideline 426. This results only in a limited number of offspring available for the different evaluations and, significantly reduces the statistical power and reduces the meaningful evaluation of the effect of the test item. In addition, at least three dose levels should be tested, and a descending sequence of dose levels should be selected with a view to demonstrating any dose-related response and a No-Observed-Adverse Effect level (NOAEL), as recommended by the OECD guideline 426. The study was conducted only with one high dose level and was not designed to establish a dose-response relationship or a no effect level. Also, according to the OECD guideline 426, on or before postnatal day 4, the size of each litter should be adjusted by eliminating extra pups to yield a uniform litter size for all litters and litter size should not exceed the average litter size for the strain of rodents used. The litter should have, as nearly as possible, equal number of male and female pups. In this publication, no standardisation of litters was apparently carried out and there is no information on the number of male and female pups per litter. Interestingly, the study started with 10 pregnant females, but later on in the publication the authors mentioned 6 to 8 litters only. There is no information on dead dams or loss of complete litter by the dams, which precludes the possibility to make any assumption on the effect of the test substance on maternal and foetal mortality. There is no information on maternal toxicity, since clinical signs, mortality, detailed clinical observations, body weight, and food consumption were not reported. It is not plausible that the dams did not show any signs of toxicity after oral treatment with such a high dose (500 mg/kg bw zinc oxide nanoparticles). In comparison, zinc oxide nanoparticles at a dose of 300 mg/kg bw was highly toxic for the pregnant mice preventing evaluation in the study described by Bara et al. 2018. Therefore, it is impossible to preclude that the observed effects in the offspring were caused by maternal toxicity. According to the OECD guideline 426, clinical signs, mortality, and detailed clinical observations should be obtained for the offspring. These observations were not reported. Body weight should be obtained weekly during the pre-weaning period and a least every two weeks post-weaning. However, in the current study body weight was only determined on postnatal day 2. Furthermore, learning and memory should be examined post weaning (e.g. 25 ± 2 days) and young adults (postnatal day 60) according to the OECD guideline 426. The animals were investigated only as young adults (postnatal day 60). Therefore, it is impossible to concluded, if anything else besides the test item influenced the learning ability, since no information is available from an earlier age. Therefore, the study is considered not reliable (RL=3).

Kumar et al. 1976 conducted a study to determine the effect of zinc supplementation on the number of implantation sites and resorptions in pregnant rats. The control group consisting of 12 pregnant females was maintained on 10 % vegetable protein diet (containing 30 ppm zinc) from Day 1 through Day 18 of pregnancy. The experimental group consisting of 13 animals was also maintained on the same diet, but received additionally 150 ppm zinc as a 2% zinc sulphate solution administered daily orally. All the animals were sacrificed on Day 18 of pregnancy, and their uteri examined for implantation sites and resorptions. Of a total number of 101 implantation sites in the 12 control animals there were two resorptions, one in each of two animals. In marked contrast, in the 13 zinc supplemented animals, there were 11 resorptions out of 116 implantations. Eight of the animals had at least one resorption each. This difference was statistically significant. The result indicates that oral administration of moderately high levels of zinc (150 ppm) may be associated with harmful effects in the course of pregnancy of rat. Due to significant deficiencies in study design and reporting, the reference is considered not reliable (RL=3).

 

Inhalation
In a pre-natal developmental toxicity study in rats, animals were exposed to nano zinc oxide (Z-Cote HP1) at concentrations of 0.3, 1.5, 7.5 mg/m³. The study was conducted in accordance with OECD 414 and under GLP. Under the conditions of this prenatal developmental toxicity study, the inhalative administration of Z-Cote HP1 to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 7.5 mg/m³ caused moderate alveolar lipoproteinosis and slight inflammation. These histopathologic findings are regarded to be adverse in nature. The relevance for humans however is not clear. In conclusion, the no observed adverse effect concentration (NOAEC) for maternal toxicity is1.5 mg/m³. The no observed adverse effect concentration (NOAEC) for prenatal developmental toxicity is 7.5 mg/m³.There were no adverse foetal findings evident at any dose.

 

non-physiological routes of exposure
Lee et al. (2016) investigated the effects of zinc oxide nanoparticles on dams and foetuses. Pregnant female rats (20 -24 rats/dose group) were dosed intravenously with the test item in 5 % glucose solution at dose levels of 5, 10, and 20 mg/kg bw/day. Administration was once daily on gestation days 6 to 20. A vehicle control group was run concurrently.
During the observations of the dams, there were no test item-related effects observed for clinical signs and food consumption as well as for. However, two females rats died at the 20 mg/kg bw/day dose level during the treatment period. Furthermore, body weight gain was decreased at the 20 mg/kg bw/day dose level. In addition, total body weight, corrected terminal body weight and net body weight changes were decreased in all treatment groups compared to the control group (statistically significantly decreased in high dose group only). During the haematological analysis, test item-related effects were observed in the dams. Total red blood cell count, haemoglobin, and haematocrit were statistically significantly decreased in all treatment groups. In addition, mean corpuscular haemoglobin concentration was statistically significantly decreased at the 10 and 20 mg/kg/day dose levels and white blood cells, neutrophils, monocytes and large unstained cells were significantly increased at the mid and high dose levels. Fibrinogen was increased in the mid and high dose groups compared to the control. Lastly, mean corpuscular haemoglobin was statistically significantly decreased at the 20 mg/kg bw/day dose level and red cell distribution width as well as haemoglobin distribution width were significantly increased. Also, during the biochemical analysis, test item-related effects were reported for the dams. Alkaline phosphatase was significantly increased in all treatment groups. Furthermore, albumin, total bilirubin and phospholipid were decreased at the 10 and 20 mg/kg bw/day dose levels. Lastly, creatinine phosphokinase was significantly decreased, and inorganic phosphorus was significantly increased at the 20 mg/kg bw/day dose level.
Histopathological abnormalities were found in the kidney, liver and lung of dams after treatment with zinc oxide nanoparticles at the 20 mg/kg bw/day dose level. Tubular dilation with basophilic change was observed in kidney, extramedullary hemopoiesis was observed in liver, and multifocal mixed cell infiltration with neutrophils, histocytes, and cellular debris was observed in lungs of treated rats. In addition, zinc level was significantly elevated in the liver, lung, and kidney of dams receiving 20 mg/kg bw/day of the test item. Lastly, no test item-related effects were noted on the reproductive parameters (number of abortions, early and late resorptions, dead foetuses, pre-implantation loss, corpora lutea and implantations), except for a statistically significant increase in number of total dead and post-implantation loss at the 20 mg/kg bw/day dose level. During the examination of foetuses, no test item-related effects were observed on sex ratio, external examinations, skeletal examinations, and visceral examinations. However, the body weight of foetus from rats treated with 20 mg/kg bw/day of the test item was significantly lower than that of the control group. In addition, the placental weight was significantly increased in female foetuses and zinc level was elevated in the liver of the foetuses. In conclusion, the LOAEL for maternal toxicity was considered to be 5 mg/kg bw/day based on the haematology and clinical chemistry findings. The NOAEL for developmental toxicity was considered to be 10 mg/kg bw/day based on the foetal death, post-implantation loss and foetal weight. No evidence of teratogenicity was observed.
The study shows experimental and reporting deficiencies as follows: Firstly, the test substance was insufficiently described (purity and stability were missing) and intravenous injection is a non-relevant route of administration for human hazard assessment. According to the OECD guideline 414, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (NOAEL) at the lowest dose level. However, a NOAEL for maternal toxicity could not be determined in the present study, since treatment-related effects were observed in all three dose groups. In addition, the body weight was not reported at the start of the study, as requested by the OECD guideline 414. In addition, it is unclear, if the uterine weight was determined together with the cervix and a macroscopical examination was carried out, which is foreseen by the OECD guideline. Lastly, individual and historical control data were missing. Since the individual and historical control data are missing, it is impossible to see if there were any outliner in the data or if the results were within the normal biological variation. Therefore, the study is considered not reliable (RL=3).

 

Conclusion
The two developmental toxicity studies according to OECD guideline 414 and GLP conducted by the group of Hong and co-workers (2014a and 2014b) are almost identical with one difference that either positively or negatively charged ZnO NP (0, 100, 200, 400 mg/kg bw/d) were administered to pregnant SD female rats during gestation. Based on the results of the study with positively charged ZnOSM20(+), a dosage level of 200 mg/kg bw/day was considered the NOAEL for both maternal toxicity and embryo–foetal development. While the study with negatively charged ZnO NPs revealed that 100 mg/kg bw/day represents the NOAEL for maternal toxicity, and the highest dose level of 400 mg/kg bw/day was considered the NOAEL for embryo–foetal development.
A non-guideline developmental toxicity study is available with evaluation of the effects of ZnO nanoparticles in comparison to bulk ZnO NP but is regarded as only of supportive nature and not reliable [RL=3] in the context of regulatory developmental toxicity. In addition, 4 publications (Hong et al 2014a and 2014b, Lee et al. 2016, Jo et al. 2013) conducted according to OECD guidelines (OECD 414 or OECD 421) were identified with two studies (Hong et al 2014a and 2014b) regarded as reliable with restrictions [RL=2] and two as not reliable (RL=3] because of either the route of administration (Lee et al. 2016) or the dose level (single high dose) chosen (Jo et al. 2013).
The results of the developmental toxicity study in pregnant SD rats conducted via an intravenous route of administration can generally be regarded as reliable with restrictions, because the study was conducted based on the OECD guideline 414 (2001) and sounds scientifically valid, but because of the non-relevant intravenous route of exposure it is only of supportive nature and overall regarded as not reliable. The NOAEL for developmental toxicity was set at 10 mg/kg bw with only some general effects on foetal mortality at a clear maternal toxic dose level (Lee et al. 2016).
In addition, in a reproductive and development toxicity screening study (non-GLP) in rats conducted according to OECD guideline 421 (Jo et al. 2013), the effects of oral (gavage) exposure of male (2 weeks before mating) and female rats (2 weeks before mating to postnatal day 4) to one high dose of ZnO NPs of 500 mg/kg bw/d was evaluated. In this study in rats treated with ZnO NPs, 3 male rats died but only a very few signs of maternal toxicity were seen and no effects on fertility and pregnancy outcome were observed, but the results showed effects on the offspring by a reduced number of born/live pups, decreased body weights of pups and increased foetal resorption. In contrast to the study summarised next (Feng Xiaoli et al. 2017), there was no difference in zinc content in the brain of treated and control pups.
The effects of a very high dose of ZnO nanoparticles (500 mg/kg bw) was investigated in rats by oral gavage daily for 18 consecutive days (on GDs 2–19) in another non-regulatory developmental neuro-toxicity study (Feng Xiaoli et al. 2017). The results showed a significantly elevated concentration of zinc in offspring brains, abnormal neuron ultra-structures, decreased proliferation and higher apoptotic death in brains of offspring. In addition, adult female offspring exhibited impaired learning and memory behaviour in the Morris water maze test. However, this study was conducted only with a very high dose level and although no maternal toxicity is reported in the publication, it is supposed that this high dose level resulted in clear maternal toxicity, based on the evidence from other animals studies with oral exposure. ZnO NPs at a dose of 300 mg/kg BW was highly toxic for the pregnant mice preventing evaluation in the study described by Bara et al. 2018.
In conclusion, the lowest NOAEL for developmental toxicity obtained from reliable developmental toxicity studies with ZnO NPs in SD rats was obtained in the study with positively charged ZnO NPs at a dose of 200 mg/kg bw/day. This dose level represents also the NOAEL for maternal toxicity.

 

 

 

Animal studies – zinc deficiency

Zinc deficiency
In total, nine non-regulatory developmental toxicity studies, the effects of zinc deficiency on various developmental endpoints were identified and investigated. All studies are regarded as only of supportive nature and not reliable [RL=3] to address developmental toxicity in a regulatory context.
The effects of zinc deficiency on brain development (Chowanadisai et al. 2005), was investigated in SD rats receiving diets containing 25 µg Zn/g diet (control), 10 µg Zn/g diet (marginally zinc deficient, MZD), or 7 µg Zn/g diet (zinc deficient, ZD) as zinc carbonate from premating to weaning. It was shown that Zinc deficiency reduces NMDA receptor expression in both brain and hippocampus areas during neonatal development, and that NMDA receptor expression remains low in adult rats following zinc repletion.
Foetal heart malformations caused by zinc deficiency of dams (Lopez et al. 2008) was investigated in SD rats receiving either a zinc-adequate (ZnA: 25 μg Zn/g) or a Zn-deficient (ZnD: <1.0 μg Zn/g) diet from GD0.5 to 4.5. It could be demonstrated that maternal zinc deficiency results in abnormal heart development. An effects of ZN deficiency on resorptions and malformed foetuses was also shown.
In 4 publications by the group of Tomat et al. (2008, 2010. 2013a and 2013 b), the effects of moderate zinc deficiency was evaluated in pregnant Wistar rats fed a zinc-deficient diet (8 mg/kg) vs a control zinc diet (30 mg/kg) during pregnancy and lactation periods and/or postweaning growth. The effects on systolic blood pressure, renal function and morphology, vascular and renal NO system and alterations in plasma lipid profile as well cardiac morphology and dysfunction in offspring and/or adult animals are described. In all studies, Zn deficiency of mother revealed an effect on the parameters investigated in the offspring during post-natal live and adulthood. It was also demonstrated that the mean birth weight of litters in Zn-restricted dams was significantly lower compared to the control groups, and that Zn-deficiency of dams resulted significantly smaller offspring with lower mean body lengths (crown–rump length), tail and head length, but not in head width.
The effects prenatal Zn deficiency (0.5–1.5 ppm Zn) in comparison to control diet (63 mg/kg Zn and 284.11 mg/kg Fe), on physical growth parameters (body weight, body length, tail length, and head length) was evaluated in pregnant Wistar rats (Shabazi et al. 2008). There was a significant difference in the physical growth indexes (body weight, body length, tail length, and head length) between the ZnD group compared to the control group.
In two studies with Zn deficiency (7 mg/kg bw) in comparison to an Zn adequate control diet (25 mg Zn/kg) fed from 3 weeks pre-conception to 3 weeks post partum, rat offspring were either subjected to insulin and glucose tolerance tests at week 5 and 10 of age (Jou et al. 2010), or the growth and glucose homeostasis (Jou et al. 2013) in offspring with either adequate or inadequate nutrition post weaning was investigated. The results showed that maternal Zn-deficiency resulted in greater serum IGF-1 concentrations and the increased postnatal weight gain in their offspring as well as impaired subsequent glucose sensitivity. It was associated with gender-specific alterations in the serum leptin concentration and the insulin signaling pathway. Offspring of the Zn-deficient mothers reacted to experimental diets, depending on their level of postnatal nutrition (excess nutrition (EN), adequate nutrition (AN), or inadequate nutrition (IN). In contrast to rats delivered from control mothers, those born to Zn-restricted dams and fed to excess during the early postnatal period went on to develop insulin resistance as young adults.

 

 

Zinc deficiency versus Zinc supplementation
The effects of zinc deficiency in comparison to zinc adequate and zinc supplemented diet on liver gene expression was investigated in a mechanistic non-regulatory study in mice (Kurita et al. 2013), and the effects of Zn supplementation following a Zn-deficient diet were evaluated in another non-regulatory study in rats (Yu et al. 2013). In addition, the effects of pre- or postnatal zinc supplementation on the immune system was investigated in a mechanistic developmental toxicity study in rats (Sharkar et al. 2011), and a comparison of diets supplemented with of two different zinc sources was studied in pigs (Payne et al. 2006). All studies were regarded as only of supportive nature and not reliable [RL=3] to address developmental toxicity in a regulatory context.
In a mechanistic study in mice (Kurita et al. 2013), the effect on gene expression of metallothionine in offspring livers was investigated when pregnant mice were fed either Zn supplemented (50 μg Zn/g diet), low-Zn diet (5.0 μg Zn/g) or control diet (35 μg Zn/g) from GD7 to delivery. Elevated gene expression was shown in prenatally Zn-deficient offspring after a single Cd administration at an age of 5 weeks.
Developmental neuro-toxicity was investigated by Yu et al. 2013 in rats fed either control (25 µg/g of diet) or a Zn-deficient (2 µg/g diet) diet during pregnancy and lactation. During post-natal life, offspring received either control or Zn-deficient diets as before, while one Zn-deficient group received control diet for post-natal Zn supplementation. It was demonstrated that pre-natal Zn-deficiency lowered birth weight and growth retardation and affected the ability to perform in the Morris Water Maze test and caused neuronal morphologic changes. Effects could be abolished, at least partly, by Zn supplementation during the post-natal period.
The effects of prenatal zinc supplementation in comparison to post partum zinc supplementation on development of the immune system of offspring was investigated by Sharkar et al. 2011. Pregnant SD rats were fed Zn-adequate diet (25 mg Zn/kg) throughout pregnancy and one group was supplemented with Zn (1.5 mg Zn in 10% sucrose) 3 times/wk. After birth, pups were subjected to a cross-fostering study design with pups of Zn-supplemented dams that continued with their biological mothers (Zn-Zn); Zn-P, pups from Zn-supplemented dams cross-fostered to placebo supplemented dams; P-P, pups of placebo-supplemented dams that continued with their biological mothers; and P-Zn, pups from placebo-supplemented dams cross-fostered to Zn-supplemented dams. It could be demonstrated that prenatal Zn supplementation suppressed antigen-specific proliferation, antibody responses and antigen presenting cell activity, whereas postnatal exposure may suppress the mucosal immune reservoir.
In a non-regulatory developmental toxicity study in pigs (Payne et al. 2006), the effects of organic and inorganic zinc (100 ppm) supplementation in the diet of sows fed from GD15 to end of lactation on the growth and intestinal morphology of offspring were compared. The results suggest that 100 ppm Zn in trace mineral premixes provide adequate Zn for an optimal growth performance of nursery pigs, but that 100 ppm additional Zn from ZnAA (ZnAA complex) in sow diets may increase pigs born or weaned per litter.
In conclusion, the newly evaluated studies support the evidence of a negative effects of prenatal zinc deficiency (range: <1 to 10 µg Zn/kg diet) in comparison to Zn adequate diets (range: 25-63 mg/kg diet) in laboratory animals on the in utero development, post-natal and adult live of offspring. In general, Zn-deficiency during pre- and post-natal life resulted in lower birth weight, growth retardation and abnormalities as well as effect on brain, heart and renal development and function. Evidence was shown that post-natal Zn supplementation may diminish these effects. It seems that pre-natal Zn supplementation may positively affect the outcome of pregnancy in sows, however, prenatal Zn supplementation may affect the immune system of rat offspring.
The newly evaluated non-regulatory and regulatory developmental toxicity studies do not change the NOAEL for developmental toxicity of 50 mg/kg bw/day obtained in rats as presented in the IUCLID endpoint summary (2014). The NOAEL was set on the basis of prenatal toxicity studies conducted with soluble zinc sulphate and zinc chloride and slightly soluble zinc carbonate in rats, mice, hamsters or rabbits via the oral route. Based on the newly evaluated studies with ZnO NPs, a higher NOAEL of 200 mg/kg bw was obtained for positively charged NPs and was already higher for negatively charged ZnO NPs.

 

 

Animal studies – others
In the non-guideline developmental toxicity study (non-GLP), the effects of increasing dietary zinc concentrations on cognitive impairments of offspring caused by lipopolysaccharide (LPS) administration early in pregnancy was investigated in female C57BL6 mice (Coyle et al. 2009).
Pregnant C57BL6 mice (Institute of Medical and Veterinary Science (IMVS) Animal Care Facility, Adelaide, SA, Australia) were treated from GD0 to parturition to one of two dietary groups and fed either a normal Zn diet (AIN-93G + 35 mg Zn/kg) or diet supplemented with Zn (AIN-93G + 100 mg Zn/kg as zinc sulfate). On GD8, mice from both dietary groups were subcutaneously injected either with LPS (0.30 mg/kg) in saline (0.85%,w/v NaCl) or saline alone. From GD 19, cages were inspected twice daily for pups. The date of birth was noted as postnatal day (PD) 1 and litter size was recorded. Pups were not handled until PD 3 to minimise cannibalism by the dams at which stage each litter was culled to a maximum of 7 pups. Pups were weaned on PD21 and housed according to gender, litter and treatment until behavioural testing. A separate cohort of pregnant mice administered the same treatments as described above were killed on GD12 (for microarray analysis of foetal brain) and GD18, and the number of resorptions and foetuses was determined.

All offspring were physically examined at PD35. Twelve male and 12 female adult offspring were randomly selected per treatment group and their weights recorded. No more than one male and one female were selected from the same litter. Visual and olfactory tests were then performed. No visual or olfactory impairments were observed in the cohort of mice randomly selected. Mice were handled every day until the beginning of testing, when offspring were assessed for impairments in spatial learning and memory and object recognition memory. Spatial learning and memory were assessed by using a cross-maze escape task. Mice were assessed on their long-term retention of the escape platform location which was placed in the same position as during the learning phase. Memory was tested 14 and 28 days (PD65 and 79, respectively) after the final day of learning and consisted of a single day of 6 trials as described for the learning period. The modified object recognition task (ORT), a specific form of episodic memory which takes advantage of the natural affinity of mice for novelty, was conducted on PD 85/86 in the same cohort of mice tested for spatial learning and memory
There were no differences in maternal weights between treatments on GD14, PD3 or PD21. There was a treatment effect on litter size at birth [p=0.000] and a significant diet×treatment interaction [p=0.042], where the litter size of the LPS group was smaller compared to all other treatments and the LPS + Zn group smaller than the Control and Control + Zn. The litter size in Control + Zn was also lower compared to Control. There was a treatment effect on pup weight at PD3 [p=0.007] in saline control compared to LPS, but no significant diet×treatment interaction. There was no difference in the number of resorptions and/or foetuses between treatment groups on GD12. However, on GD18 there was a significant diet×treatment interaction [p=0.021] in the number of foetuses and in the total number of resorptions + foetuses [p<0.006], where there was significantly fewer resorptions + foetuses in the LPS group alone, than all other treatment groups. There was no effect of treatment, diet or sex on escape latency and no treatment×diet interaction on spatial learning. When memory was assessed at 14 days after learning the task, there were no treatment, diet or sex effects and no treatment×diet interactions for escape latency. When the mice were re-tested at 28 days after learning, there were also no treatment or diet effects and no treatment×diet interactions. In the object recognition task, the mice from each treatment did not demonstrate a preference for either side of the box during the sample phase where two identical objects were presented. In the choice phase, where a novel and familiar object was presented, there was a significant treatment effect [p=0.005] on total exploration time (T2) where saline-treated groups had longer times than those with LPS). There was a significant treatment effect [p=0.0097] on the h1 index, a measure of difference in exploratory behaviour between the sample and choice phase, here, saline-treated groups had a significantly lower h1 index than those with LPS. There was a significant treatment effect on object recognition performance [p=0.0008], where the d2 index (relative discrimination index) was significantly lower with LPS than saline (=0.0008). There was also a significant diet effect [p=0.0025], where groups with normal zinc diet had a lower index than with supplemented zinc (p=0.0025). There was a strong treatment×diet interaction [p<0.0001], where the LPS group was significantly (p<0.001) lower than all other groups. No changes in gene expression (≥2-fold) were identified in comparisons between Control versus LPS, Control versus Control + Zn, LPS versus LPS + Zn or Control + Zn versus LPS + Zn for the 21,587 unique mouse genes. Six genes were identified that were the most uniformly changed in colour and spot intensity and are of interest because they are all known to influence proliferation and differentiation of neuronal tissue. All six genes were down-regulated by LPS relative to the controls.
Prenatal LPS administration resulted in reduced litter size at birth and a reduction in the number of live foetuses in utero at GD18, which was not apparent at GD12. A protective influence of dietary zinc treatment was shown. Dietary zinc supplementation prevented the LPS-mediated reduction in foetal numbers at GD18, but did not provide complete protection at birth, where the LPS + Zn group had higher number of foetuses per litter compared to LPS alone but a smaller litter size compared with controls. No influence of prenatal LPS on spatial memory could be demonstrated in the water cross-maze escape task either at 14 or 28 days after the training phase. It could be demonstrated that LPS administration in early pregnancy can cause aberrant behaviour in adult offspring in an object recognition task. The LPS-treated mice spent more time exploring the familiar object whereas all other treatment groups spent the majority of their time exploring the novel object. Dietary zinc supplementation throughout pregnancy can nullify this LPS-mediated anomaly. The LPS + Zn group performed to the same levels as the controls in the object recognition task.
This well conducted and documented non-regulatory developmental toxicity study in mice supports the evidence of a protective effect of dietary zinc supplementation (100 mg/kg diet) during pregnancy in a non-lethal LPS model. Nevertheless, although the study was conducted by a relevant route of exposure and the overall quality seems to be appropriate it can only be regarded as of supportive nature, because the LPS model represents an unsuitable non-standard system for evaluation of developmental toxicity [RL=3]. The study is included for information purposes only.

In this non-regulatory developmental toxicity study (non-GLP), the effects of increasing or decreasing dietary zinc concentrations on the teratogenic effects of LPS was investigated in female C57BL6 mice (Chua et al. 2006). Pregnant C57BL6 mice (Institute of Medical and Veterinary Science (IMVS) Animal Care Facility, Adelaide, SA, Australia) were randomly allocated to different treatment groups on GD1 and fed specially formulated diets containing low Zn (5 mg/kg), normal Zn (35 mg/kg), or supplemented Zn (100 mg/kg as zinc sulfate) throughout their pregnancy.
Mice were randomly allocated into six different groups where they were either treated with LPS or saline (S), and fed diets containing low (LPS+Zn5 / S+Zn5), normal (LPS+Zn35 / S+Zn35), or supplemented Zn (LPS+Zn100 / S+Zn100) from GD1 throughout pregnancy. On GD8, mice in the LPS group were injected subcutaneously with 0.5 µg/g body weight LPS in 0.85% saline. Control mice were treated with 0.85% saline in a similar fashion. Mice were killed on GD18 and uteri were immediately excised, weighed, and examined for number of resorption sites. Individual foetuses were separated from the placentas, weighed, and crown-rump length was measured. Foetuses were examined under low power magnification to determine the extent of physical abnormalities such as microphthalmia, anophthalmia, cleft lip, micrognathia, microcephaly, exencephaly, and other obvious malformations.
There were no differences in the percentage of successful pregnancies between LPS-treated mice fed 5, 35, or 100 mg/kg Zn diet. However, saline-treated mice fed the low-Zn(5 mg/kg) diet had the lowest pregnancy success (55%) compared with peers fed the normal (35 mg/kg) or supplemented (100 mg/kg) Zn diet (88% and 64%, respectively). The normal success rate as assessed in our laboratory over the years is between 80 and 90%. Only the saline-treated mice on normal Zn diet fell within this category. LPS combined with Zn deficiency had a more severe effect on the foetus than LPS or saline combined with normal or supplemented Zn. There were more resorption sites in LPS-treated dams than saline control dams regardless of dietary Zn consumption. LPS dams had 26–57% resorptions per litter compared with 7–27% in saline controls. Although LPS dams on the low and supplemented Zn diets had slightly more resorptions than those fed the normal Zn diet, this observation was not significantly different. However, LPS dams on the normal Zn diet had significantly more resorptions than the saline dams on the same diet (22% versus 7%, respectively).
Foetal weights were lower in the LPS+Zn5 and LPS+Zn35 groups compared with LPS+Zn100 group and saline+35Zn foetuses (p<0.001), respectively. However, there was no difference in foetal weights between saline control foetuses regardless of dietary Zn consumption. LPS-Zn supplemented dams had larger foetuses (in terms of crown-rump length) compared with those from LPS+Zn 35 group. There was no difference in size between LPS+Zn5 and LPS+Zn35 foetuses, although foetuses from the latter group were significantly smaller when compared with saline control foetuses exposed to the same diet (p<0.001). External malformations were most profound in foetuses from LPS+Zn5 group (96%) compared with all other treatment/dietary groups. The most common abnormalities present were anophthalmia (80%), exencephaly (60%), and microencephaly (40%). Although these abnormalities were also present in the LPS+Zn35 group, they occurred at a significantly less frequency. Foetuses on the Zn-supplemented diet were least affected by LPS, mainly exhibiting eye abnormalities commonly found in the strain of mice used. Other abnormalities observed included cleft lip, microphthalmia, micrognathia, agnathia, and spinal haemorrhage.
LPS-treated foetuses from dams fed 5 and 35 mg/kg zinc via the diet had a significantly higher number of abnormalities per litter (2- and 1- fold saline controls, respectively) compared with those from LPS+zinc supplemented dams, which were not significantly different from the saline control groups. The beneficial effect and importance of zinc was also reflected in the larger size of foetuses (weight and crown-rump length) from the LPS+zinc–supplemented treatment group.
The data of this limited developmental toxicity study in mice to evaluate the effect of dietary zinc concentrations on the outcome of a non-lethal LPS dose during pregnancy (GD8) support the evidence that dietary zinc supplementation throughout pregnancy ameliorates LPS-induced teratogenicity. The study has limitations with respect to the performance of the study, because only a low number of maternal animals (n=9) was included preventing a meaningful evaluation of the data and shows therefore significant methodological deficiencies. In addition, only a limited no. of endpoints was addressed compared to regulatory standard studies, and data were not reported in detail. Although a relevant route of exposure (oral) was chosen, the data can only be regarded as supportive and not reliable [RL=3]. The study is included for information purposes only.

 

In the present non-regulatory mechanistic developmental toxicity study, dams were treated with a single dose of zinc (ZnSO4, 2 mg/kg, subcutaneously) in an attempt to prevent or ameliorate the impairments induced by prenatal LPS exposure (Galvão et al. 2015).
Fifteen pregnant Wistar rats (12–13 weeks, 226–266 g), were used. The dams were randomly divided into three groups (n=5 per group). On postnatal day (PND)81–86), two or three female offspring from each litter were used for adult offspring evaluation (n=10–12 per group). The male offspring were separated for use in other experiments. Lipopolysaccharide (LPS) was administered intraperitoneally (i.p.) to pregnant dams at 100 μg/kg on GD9.5. One hour after LPS exposure, the dams also received a single dose of sterile saline (0.9% NaCl, 0.2 ml/100 g) or zinc (ZnSO4; 2 mg/kg) subcutaneously (s.c.). The control group consisted of pregnant rats that received only sterile saline on GD 9.5 (0.2 ml/100 g, i.p.) and an additional saline injection after 1 h (0.2 ml/100 g, s.c.). Female animals in dioestrus or metestrus were subjected to acute restrain stress considered as a simple and painless model of stress that does not cause lasting impairments for a 2-h session which was considered sufficient to activate the HPA axis, increasing circulating corticosterone levels. In the final 5 min of restraint (i.e., after 115min of restraint), the rats were observed for 22-kHz ultrasonic vocalizations in the restraint tube. The automatically recorded parameters during the 5-min session included the number of vocalizations, mean vocalization duration time, maximal vocalization duration, minimal vocalization duration, total silence duration, mean silence duration interval, maximal silence duration interval, and minimal silence duration interval. The durations were recorded in seconds.
Immediately after the ultrasonic vocalization test, the rats were placed and observed in an open field to evaluate exploratory/motor and anxiety parameters. The following parameters were manually or automatically recorded over a period of 5 min: distance travelled (cm), average velocity (cm/s), rearing frequency, self-grooming (s), and time spent in the central, intermediate, and peripheral zones (s). Immediately after the open field test, trunk blood was collected serum corticosterone levels were determined in duplicate using an enzyme-linked immunosorbent assay. The same serum samples were used to determine BDNF levels by an enzyme-linked immunosorbent assay. Monoamine and monoamine metabolite levels in the hypothalamus and striatum were measured by using high-performance liquid chromatography (HPLC) in the same rats that were behaviourally evaluated and had their serum collected. Prenatal zinc administration increased the maximal silence intervals in offspring that were exposed to LPS during gestation compared with the LPS and saline groups after acute restraint stress (p=0.0032). The rats that were prenatally treated with zinc responded less in the stress vocalization test, although no differences in the number of vocalizations were found among the three groups (p=0.5841). Prenatal zinc increased the distance travelled (p=0.0010) and average velocity (p=0.0010) in offspring that were exposed to LPS during gestation compared with the LPS and saline groups after acute restraint stress. Self-grooming was decreased in the LPS + Zn group (p=0.0004) compared with the LPS and saline groups, but rearing frequency (p=0.9718) and anxiety parameters were statistically the same among the three groups (p<0.05). Serum analyses revealed that prenatal treatment with zinc reduced corticosterone levels in offspring exposed to LPS during gestation compared with offspring in the LPS + SAL group after acute restraint stress (p=0.0225). BDNF levels were statistically the same among the three groups (p=0.4565). Offspring that received prenatal zinc after LPS exhibited a reduction of striatal norepinephrine metabolite levels (p=0.0026) compared with rats exposed to LPS and saline during gestation. Norepinephrine turnover was reduced in the LPS + Zn group compared with the SAL + SAL group (p=0.0384). The other neurotransmitter and metabolite levels and turnover in the striatum and hypothalamus were statistically the same among the three groups (p<0.05)
The data show that prenatal zinc exposure (ZnSO4, 2 mg/kg, s.c.) 1 hour after LPS treatment (100 g/kg, i.p.) on GD9.5 reduced the acute restraint stress response in adult rat offspring on PND81-86. There was evidence of a lower stress response, reflected by behavioural and neuroendocrine parameters, including longer periods of silence in the 22-kHz ultrasonic vocalization assessment, increased locomotor activity, decreased self-grooming, and reduced serum corticosterone and striatal norepinephrine turnover. The findings suggest a potential beneficial effect of prenatal zinc exposure after LPS, in which the adult stress response is reduced in offspring that are stricken by infectious or inflammatory processes during gestation. Thus, maternal zinc treatment during gestation may be beneficial.
The results of the study support the evidence of a beneficial effect of prenatal zinc exposure on the stress response in adult rats in a non-lethal LPS model. Nevertheless, the study was conducted according to an unsuitable test system for evaluation of the endpoint developmental toxicity. It represents a non-lethal LPS model in pregnant rats with only one single administration of ZnSO4 shortly after LPS administration according to a non-relevant route of exposure (s.c.). Therefore, although the study seems to be appropriately conducted and the results adequately reported it is only of supportive nature and regarded as not reliable [RL=3]. The study is included for information purposes only and is not contained in an endpoint study record due to lack of relevance.

This non-regulatory mechanistic developmental toxicity study (non-GLP) with dietary administration was conducted in adult hens to compare the effects of ZnO NPs with ZnSO4 treatment on livers in F1 chicken (Hao et al. 2007). Test material: ZnO NPs (Beijing DK Nano Technology Co. LTD, Beijing, China), Particle size: 30 nm, Surface area: 50 m2/g, Density: 5.606 g/cm³; ZnSO4 (no details) Hens (Jinghong-1 strain) received the experimental diets from 6 wks to 30 wks of age. The two treatments were ZnSO4-200 mg/kg and ZnO-NP-200 mg/kg of diet (equivalent to about 20 mg/kg bw) representing a common inclusion rate in hen diets. A total of 400 pullets were randomly assigned to the two treatments, with five replicates per treatment and forty animals per replicate. After 24 wks treatments, the hens were artificially inseminated with fresh semen. Eggs were collected and stored in incubators. After hatching, the F1 chickens were raised under same conditions on the same diet (no additional ZnO NPs treatment for F1 animals). Liver samples were collected at embryonic day 18 (E-18), postnatal day 3 (d-3), postnatal day 5 (d-5), postnatal day 10 (d-10) and postnatal day 20 (d-20) and the liver samples were frozen immediately in liquid nitrogen for further analysis (6 animals/group). Part of the liver samples were processed for histopathology. Liver samples were analysed for mRNA by real-time quantitative RT-PCR and Western blotting was conducted.
Histopathologic evaluations showed that in the ZnSO4-200 mg/kg treatment group, hepatocellular cords and the shape of hepatocytes were regular from E-18 to d-20. In the ZnO NP treatment group, the hepatocellular cords were also regular; however, infiltrated inflammatory cells were observed on d-20, liver hepatocytes had irregular organization with lesions from d-5 and the affected hepatocytes had pyknotic nuclei on d-20. Abnormal gene expression and protein levels of lipid synthesis enzymes due to ZnO NPs were observed in F1 chicken livers, and the gene expression and protein levels of growth-related factors were altered. Cell damage or apoptosis was induced in the ZnO NP group F1 chicken livers.
This investigation explored the impacts of ZnO NPs on offspring liver function at the molecular level of gene and protein expression after maternal oral exposure. Three pathways were investigated: lipid synthesis, growth related factors and cell toxic biomarkers/apoptosis at 5 different time points from E 18 to d-20. It was found that the expression of 15, 16, and 16 genes in lipid synthesis, growth related factors and cell toxic biomarkers/apoptosis signalling pathway respectively in F1 animal liver were altered by ZnO NPs compared to ZnSO4. The proteins in these signalling pathways (five in each pathways analyzed) in F1 animal liver were also changed by ZnO NPs compared to ZnSO4. The results suggest that ZnO NPs could be toxic on offspring liver development, mainly influencing lipid synthesis, growth, and lesions or apoptosis.
In this mechanistic developmental toxicity study, the effects of ZnO NPs in comparison to ZnSO4 was evaluated in chicken livers from hens exposed to the test material prior to and during insemination and egg production. The study was conducted according to an unsuitable test system for evaluation of the endpoint developmental toxicity. The animals used are not recommended by regulatory guidelines and the endpoint addressed (liver toxicity) is more related to general toxicity. Standard developmental toxicity parameters were not addressed in the study. In addition, the study is a comparative study with two forms of Zn but without control. Therefore, although the study seems to be appropriately conducted and the results adequately reported it is only of supportive nature and not reliable [RL=3]. The study is included for information purposes only and is not contained in an endpoint study record due to lack of relevance.

In this mechanistic non-regulatory developmental toxicity study (non-GLP) in mice, it was investigated whether dietary Zn supplementation throughout pregnancy can also prevent ethanol-related dysmorphology (Summers et al. 2009). Pregnant C57BL⁄ 6J mice (Institute of Medical and Veterinary Science, IMVS, Adelaide) were assigned to 1 of 4 treatment groups: (1) saline + control diet, (2) ethanol + control diet, (3) saline + Zn supplemented diet, or (4) ethanol + Zn supplemented diet. Mice were fed the control diet (35 mg Zn⁄kg) or the Zn-supplemented diet (200 mg Zn⁄kg) from GD 0 to GD 18. Pregnant mice received 2 intraperitoneal injections, separated by 4 hours, of either saline (0.85% w⁄v NaCl) or 25% ethanol (v⁄v) in saline solution at a dose of 2.9 g⁄kg (0.015 ml⁄g body weight). Blood samples were collected for maternal plasma Zn analysis. Foetuses (n=11/group) from the saline, saline + Zn, ethanol and ethanol + Zn groups were assessed for external birth abnormalities on GD 18. Individual foetuses and their placentas were then separated and weighed and the foetal crown-rump length (CRL) was measured. Foetuses were examined for external abnormalities (e.g., anophthalmia, microphthalmia, micrognathia, limb defects, haemorrhaging). As microcephaly (small head for body size) is a common feature of foetal alcohol syndrome, head dimensions [height (h), width (w), and depth (d)] were measured to determine foetal head volume. In a separate cohort, pregnant dams were allocated to 1 of the 4 treatment groups on GD 0 (n = 13 to 16⁄ group) and were subjected to the same GD 8 ethanol or saline treatment as described above. The control or Zn-supplemented diet, however, was fed to mice from GD 0 to GD 20. Postnatal growth and survival of offspring were examined from birth until postnatal day 60. To examine the effect of dietary Zn-supplementation on the liver metallothionein, Zn and plasma Zn response following ethanol exposure, a separate group of pregnant mice were allocated to either a control diet or a Zn-supplemented diet from GD 0. On GD 8, mice were treated with ethanol.
Maternal ethanol treatment on GD 8, either by ethanol alone or with dietary Zn supplementation, had no effect on litter size or the percentage of foetal resorptions compared with saline treatment alone. In addition, there was no effect of GD 8 treatment or diet throughout pregnancy on foetal weight, placental weight, foetal CRL and foetal head volume⁄CRL ratio. Foetuses exposed to ethanol alone on GD 8 exhibited a significantly higher incidence of external abnormalities compared with saline, saline + Zn and ethanol + Zn foetuses (p<0.05). The ethanol group had the greatest number of litters affected, with 11 of 11 litters containing abnormal foetuses, compared to saline (8⁄11), saline + Zn (7⁄11), or ethanol + Zn (7⁄11). In addition, 6 of 11 of the litters from the ethanol group contained 2 or more abnormal foetuses, compared to ethanol + Zn (1⁄11), saline and saline + Zn groups (each 0⁄11). Sixty percent of the total abnormalities observed in all groups involved malformations of the eye, with microphthalmia being the most frequent of all abnormalities, followed by anophthalmia, haemorrhaging of the craniumor dorsal region and limb abnormalities. On GD 18, maternal plasma Zn concentrations revealed that both saline + Zn and ethanol + Zn dams that were supplemented with dietary Zn from GD 1-18 had higher plasma Zn concentrations (>1fold) compared to the un-supplemented dams. There was no effect of maternal ethanol treatment or dietary Zn supplementation on litter size at birth. There was also no significant treatment effect on postnatal growth of offspring [p=0.09] or treatment day interaction [p=0.12], as offspring from all groups demonstrated similar weight gain from PND 3 and 21. There were 7 stillbirths in 4 out of 16 litters in the ethanol group compared to 1 stillbirth in 15 of the litters in the ethanol + Zn group. There were no stillbirths in the saline or saline + Zn groups. Between birth and postnatal day 3, 25 pups died in the ethanol group compared with a maximum of 11 pups in the other 3 treatment groups. By PND 60, 44% of pups had died in the ethanol group (40% excluding stillbirths) and this was at least double the number of deaths in any of the other treatment groups. Of litters in the ethanol group, 69% were affected by postnatal death, compared to between 27 and 54% in the other groups. The cumulative survival of pups (100% Deaths) in the ethanol group was significantly lower than those in all other treatment groups.
In this very specific non-regulatory developmental toxicity study, foetuses from dams treated with ethanol alone in early pregnancy had a significantly greater incidence of physical abnormalities (26%) compared to those from the saline (10%), saline + Zn (9%), or ethanol + Zn (12%) groups. The incidence of abnormalities in ethanol + Zn-supplemented foetuses was not different from saline-treated foetuses. While ethanol exposure did not affect the number of foetal resorptions or pre- or postnatal weight, there were more stillbirths with ethanol alone, and cumulative postnatal mortality was significantly higher in offspring exposed to ethanol alone (35% deaths) compared to all other treatment groups (13.5 to 20.5% deaths). Mice supplemented with Zn throughout pregnancy had higher plasma Zn concentrations than those in un-supplemented groups. While dietary Zn supplementation was shown to be protective against the effects of ethanol exposure on foetal dysmorphology and postnatal mortality, there did not appear to be any benefits of excess Zn in the diet on the growth or survival of offspring in the normal pregnancy setting. No adverse effects of the high Zn diet on resorptions, foetal morphology or on postnatal survival were found.
This non-regulatory developmental toxicity study in mice supports the role of Zn in foetal dysmorphology and postnatal death caused by acute ethanol exposure. In addition, there was no effect of zinc supplementation on development of offspring under the conditions of the study. Although the study appears to be well conducted and reported it shows some limitations in the context of regulatory relevance. The number of animals subjected to the study groups was lower than recommended by guidelines and only a limited number of study endpoints as appropriate for regulatory studies were addressed. In addition, the purpose of the study was to evaluate the effect on ethanol exposure and thus only one group of Zn supplemented animals is available. The endpoints selected were specific for morphological effects of ethanol and therefore only a limited number of external malformations were investigated, and no skeletal or visceral evaluations were included in the study. Therefore, the study is only of supportive nature and regarded as not reliable [RL=3]. The study is included for information purposes only.

In this mechanistic non-guideline developmental neuro- toxicity study (non-GLP) in mice, it was investigated whether subcutaneous treatment of dams with ZnO NPs on several days during gestation affect monoaminergic neurotransmitter levels in the brain of mouse offspring (Okada et al. 2013). Test material: ZnO NPs (Mz-300, Tayca Co., Osaka, Japan), Primary diameter: 30-40 nm. ZnO NPs were dispersed in saline containing 0.05% Tween-80. Pregnant ICR mice (8-11 week at gestation day 1, SLC Co., Shizuoka, Japan) were subcutaneously treated with 100 μg/mouse/day on GD 5, 8, 11, 14 and 17 (total 500 μg/mouse). Control mice were treated with saline containing 0.05% Tween-80. In each group, pups were weaned on postnatal day 21. Brains were removed from 6-week-old anesthetized male pups (n = 8/group) and dissected to 9 regions: prefrontal cor-tex, neostriatum (caudate-putamen), nucleus accumbens, hippocampus, amygdala, hypothalamus, midbrain, brain-stem, and cerebellum, and dopamine (DA), 3, 4-dihydrox-yphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), noradrenalin (NA), normetanephrin (NM), 3-methoxy-4-hydrophenyl (MHPG), serotonin (5-HT), and 5-hydrox-yindole-3-acetic acid (5-HIAA) were analyzed and protein concentration was measured.
HPLC analysis demonstrated that DA levels were increased in hippocampus in the ZnO NP exposure group. In the sample with levels of DA, metabolites, homovanillic acid was increased in the prefrontal cortex and hippocampus, and 3, 4-dihydroxy-phenylacetic acid was increased in the prefrontal cortex by prenatal ZnO NP exposure. Furthermore, DA turnover levels were increased in the prefrontal cortex, neostriatum, nucleus accumbens, and amygdala in the ZnO NP exposure group. It was also found that changes of the levels of serotonin in the hypothalamus, and of the levels of 5-HIAA (5-HT metabolite) occurred in the prefrontal cortex and hippocampus in the ZnO NP-exposed group. The levels of 5-HT turnover were increased in each of the regions except for the cere-bellum by prenatal ZnO NP exposure. The present study indicated that prenatal exposure to ZnO NPs might disrupt the monoaminergic system of mice offspring exposed in utero with ZnO NPs.
In this mechanistic non-guideline developmental neuro-toxicity study in mice, a very specific endpoint (monoaminergic neurotransmitter levels in the brain) was evaluated which is not useful in a regulatory context. In addition, non-relevant route of exposure was chosen (sc), the treatment regimen was not guideline conform and relevant endpoint for evaluation of developmental neurotoxicity were not evaluated. Therefore, the study is only of supportive nature and regarded as not reliable [RL=3]. The study is included for information purposes only.

Justification for classification or non-classification

Neither the impairment of fertility nor the developmental toxicity of the zinc category substances is considered end-points of concern for humans. Based on the available information in experimental animals as well as in humans, there is no reason to classify any of the zinc category substances for reproductive toxicity in accordance with regulation (EC) 1272/2008.