Zinc selenite

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

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

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.