Zinc diricinoleate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for data waiving:

other:

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not applicable

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley Breeding Laboratories, Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA
- Age at study initiation: 30-35 d
- Housing: Polycarbonate cages with stainless-steel wire lids
- Diet: Rodent chow(Lab Diet, Richmond Standard, PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: Deionized water, ad libitum
- Acclimation period: 2 wk


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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

Details on mating procedure:

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

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

2 generations

Frequency of treatment:

7 d/wk

Details on study schedule:

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

Remarks:

Doses / Concentrations:
7.5, 15, 30 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes


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

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

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


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


GROSS EXAMINATION OF DEAD PUPS:
No

Postmortem examinations (parental animals):

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


HISTOPATHOLOGY / ORGAN WEIGHTS:

Organ weights: During the necropsy, organ weights were recorded for the kidneys, liver, brain, pituitary, adrenals, pancreas, thymus, spleen, testes, epididymides, prostate, and seminal vesicles. Fo male organ weights were also adjusted to body weight for statistical analysis.

Histopathology: Tissue samples collected from organs listed above for histopathologic evaluation were fixed in either Bouins solution (all reproductive tissues) or 10% neutral buffered formalin (all other tissues). After fixation, the tissue samples were trimmed, processed, embedded in paraffin, cut at 6 μm and stained with hematoxylin and eosin.

Postmortem examinations (offspring):

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

Statistics:

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

Reproductive indices:

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

Offspring viability indices:

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

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

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


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): All ZnCl2-treated F0 males experienced significant reduction in body weight after the 1st week of dosing and this trend continued up to the end of the experiment. The total weight gain of males was significantly reduced (dose dependent) in the low-, mid- and high-dose groups. The males experienced 0, 8, 20, and 12% mortality in control, low-, mid- and high-dose groups, respectively. In the F0 females, total weight gain and percent reduction in the low- mid- and high-dose groups were not significantly different from the control.


HEMATOLOGY AND CLINICAL CHEMISTRY: None of the hemogram or leukogram values of both Fo and F1 males and females among the ZnCl2-treated groups were different from those of the control groups. However, there was a trend toward decreased values of Packed Cell Volume (PCV). The clinical chemistry findings in males and females of both generations did not show any significant difference from those of their controls. However, in mid- and high-dose males of both generations, there seemed to a trend toward elevated values of Amyl, ALK, and GLu.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): In F0 rats, ZnCl2 treatment caused a significant reduction on the fertility, litter size, and the viability indices (Days 0 and 4) were significantly reduced at the high-dose group compared to control.

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


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


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


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

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING): The F1 males in the mid- and high-dose groups experienced a significant reduction in body weight after the 1st week of dosing and the low-dose group experienced a similar reduction after the 2nd week of dosing. These trends continued up to the end of the experiment. The total weight gain of F1 males was significantly reduced (dose dependent) in the low, mid-, and high-dose groups. The males experienced 0, 12, 8, and 4% mortality in the control, low-, mid- and high-dose groups, respectively. The mortality among the F1 females was 0, 8, 12, and 20% in the control, low-, mid- and high-dose groups, respectively.


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


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


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


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


HISTOPATHOLOGY (OFFSPRING): In males, the most biologically meaningful lesions were found in the reproductive system (prostatic acinar atrophy and inflammation) and the hematopoietic-lymphoreticular system (splenic lymphoid depletion and hemosiderosis and thymic atrophy) of 30.00 mg/kg/day ZnCl2-treated groups. These results indicated that ZnCl2 exposure has only mild effects on the reproductive performance of rats.

No significant changes in clinical pathology values or organ weights correlated with these lesions. None of the microscopic changes in target organs were of great magnitude. All unscheduled deaths were confined to the ZnCl2-treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the ZnCl2-treated females was similar to that seen in the males, except that no lesions were seen in the reproductive system. The correlations and biological interpretations were also very similar.


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

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

7.5 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

not specified

None

Conclusions:

Under the test conditions, administration of test material to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults and offspring at 30 and 15 mg/kg/d. Although effects were seen at 7.5 mg/kg/d, these were considered to be toxicologically non significant and is therefore considered to be the "No Observed Adverse Effect Level" (NOAEL).

Executive summary:

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

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

Under the test conditions, administration of test material to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults and offspring at 30 and 15 mg/kg/d. Although effects were seen at 7.5 mg/kg/d, these were considered to be toxicologically non significant and is therefore considered to be the "No Observed Adverse Effect Level" (NOAEL).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7.5 mg/kg bw/day

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

The reproductive toxicity of zinc compounds has been investigated in one and two generation reproductive toxicity studies in which rats or mice were dosed by gavage or via the diet with soluble zinc compounds (i.e., zinc chloride, zinc sulphate) at exposure levels up to 14 mg Zn/kg bw/day (gavage) or 200 mg Zn/kg bw/day (diet) (Khan AT, Atkinson A, Graham TC, Green M, Ali S, Thompson SJ and Shireen KF (2001) Effects of low levels of zinc on reproductive performance of rats. Environ Sci 8: 367—381; Khan AT, Atkinson A, Graham TC, Thompson SJ, Ali S and Shireen KF (2003) Effects of low levels of zinc on reproductive performance of mice. Environ Sci 10: 279—290; Khan AT, Graham TC, Ogden L, Salwa AS, Thompson SJ, Shireen KF, and Mahboob M (2007). A two-generational reproductive toxicity study of zinc in rats. J. Environ. Sci. Health Part B 42: 403–415).

Further information on potential effects of zinc compounds on male or female reproductive organs could be retrieved from subchronic toxicity studies as conducted by Maita et al. (

Maita K, Hirano M, Mitsumori K, Takashi K and Shirasu Y (1981). Subacute toxicity studies with zinc sulfate in mice and rats. J. Pesticide Sci. 6:327-336.)

and Edwards and Buckley ( Edwards K and Buckley P (1995). Study report zinc monoglycerolate: 13 week feeding study in rats. EU risk assessment report for zinc metal. Testing laboratory: Environmental Safety Laboratory, Unilever Research, Bedford, England. Report no.: FT930588).

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- US-EPA (US Environmental Protection Agency) (2005) Toxicological review of zinc and compounds. Washington, DC, USA). 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.,(

Khan AT, Atkinson A, Graham TC, Green M, Ali S, Thompson SJ and Shireen KF (2001) Effects of low levels of zinc on reproductive performance of rats. Environ Sci 8: 367—381;

Khan AT, Atkinson A, Graham TC, Thompson SJ, Ali S and Shireen KF (2003) Effects of low levels of zinc on reproductive performance of mice. Environ Sci 10: 279—290;

Khan AT, Graham TC, Ogden L, Salwa AS, Thompson SJ, Shireen KF, and Mahboob M (2007). A two-generational reproductive toxicity study of zinc in rats. J. Environ. Sci. Health Part B 42: 403–415) or, in case of the study conducted by Kumar (Kumar S (1976). Effect of zinc supplementation on rats during pregnancy. Nutrition Reports International. 13(1): 33-36. Testing laboratory: National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India). . Effect of zinc supplementation on rats during pregnancy. Nutrition Reports International. 13(1): 33-36. Testing laboratory: National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India), when other non-zinc relevant study specificities could have impacted the study outcome.

In a large number of controlled trials, dietary supplementation with zinc rate of 20 mg/day and 30 mg/day did not result in any adverse reproductive effects in healthy pregnant women as summarised in WHO (2001). Environmental Health Criteria 221 Zinc.http://www.inchem.org/documents/ehc/ehc/ehc221.htm#1 and ATSDR (Agency for Toxic Substances and Disease Registry) (2005). Toxicological profile for zinc. U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA, USA

Overview of experimental studies on fertility

Test substance	Method	Results	Remarks	Reference
Zinc chloride	One-generation study in ratsadministered zinc chloride at doses of 0, 3.6, 7.2, 14.4 mg Zn/kg bw/d in water over one generation by gavage. Exposure started 77 days prior to mating	As of 3.5 mg Zn/kg bw/day: P - Mortality↑ ; body weight gain↓; fertility indext↓; thymus atrophy F1 - litter size (non significant)↓; number of surviving pubs (non significant)↓;   As of 7.2 mg Zn/kg bw/day: P – hemosidosis of spleen; lymphocyte deficiency F1 - number of surviving pubs↓; BW gain (PND 21)↓	2 (reliable with restrictions) supporting study	Khan AT, Atkinson A, Graham TC, Green M, Ali S, Thompson SJ and Shireen KF (2001) Effects of low levels of zinc on reproductive performance of rats. Environ Sci 8: 367—381
Zinc chloride	One-generation study in miceadministered zinc chloride at doses of 0, 0.75, 1.5 and 3, mg Zn/kg bw/d respectively, 0. 1.5, 3 and 6, mg Zn/kg bw/d in water with 1.5mL HNO3/l over one generation by gavage. Exposure started 49 days prior to mating	0.75 resp. 1.5 mg Zn/kg bw/day: P- Mortality↑; body weight gain↓; abs./rel. Liver/thymus/ spleen weight↓; fertility indext↓ ; number pregnancies↓ F1- litter size (non significant)↓; number of surviving pubs (non significant)↓;   1.5 resp. 3 mg Zn/kg bw/day: P- body weight gain↓; F1– 14day survival index↓;   3 resp. 6 mg Zn/kg bw/day: F1– only 1 birth; 9 still births.	2 (reliable with restrictions) supporting study	Khan et al., 2001
Zinc chloride	Two-generation study in ratsadministered zinc chloride at doses of 7.5, 15and 30 mg/kg bw/d in water over two successive generations via the oral route. Application procedure not specified but likely oral gavage. Exposure started 77 days prior to mating.	As of 3.5 mg Zn/kg bw/day: P - Mortality↑; body weight gain↓; abs/rel liver/kidney weight↓; lesions in GI tract, inflammation in prostate F1 - Mortality↑; body weight gain↓; abs/rel brain/prostate/spleen weight↓; F2 – no effects   7.2 mg Zn/kg bw/day: P – abs./rel. brain/seminal vesicle weight↓; F1 - abs/rel liver/adrenal/seminal vesicle weight↓ F2 – no effects   14.1 mg Zn/kg bw/day: P – abs./rel. Spleen/uterus weight↓; F1 - body weight gain (PND21)↓; abs/rel kidney weight↓; litter size and #surviving pubs until PND4↓; F2 – body weight gain (PND21)↓; abs/rel kidney weight↓; litter size and number surviving pubs until PND4↓;   Maternal toxicity at any dose level. The NOAEL for fertility and development toxicity is about 15 mg ZnCl2/kg bw/d, this corresponds to 7.2 mg Zinc/kg bw/day. No NOAEL for systemic toxicity could be derived.	2 (reliable with restrictions) supporting study	Khan AT, Graham TC, Ogden L, Salwa AS, Thompson SJ, Shireen KF, and Mahboob M (2007). A two-generational reproductive toxicity study of zinc in rats. J. Environ. Sci. Health Part B 42: 403–415.
Zinc sulphate	Charles foster rats fed with a diet containing 4000ppm Zn (in form of zinc sulphate); exposure equals 200 mg Zn/kg bw exposure started 30-32 days prior to mating.	200 mg Zn/kg bw/day P – Zn-concentration in testis and sperm↑; sperm mobilityâ; number of pregnancies↓ F1 – number of live births↓	2 (reliable with restrictions) supporting study	Samanta K and Pal B (1986). Zinc feeding and fertility of male rats. Internat. J. Vit. Nutr. Res. 56: 105-107. Testing laboratory: Department of Biochemistry, School of Tropical Medicine, Calcutta, India.

Short description of key information:
A range of studies have been conducted to assess the effects of zinc on fertility and reproductive performance, most of them with very soluble zinc chloride and zinc sulphate. A complete overview and review of available fertility studies is available in the EU risk assessment of zinc compounds (EU RAR, 2008), the review of the of health effects of zinc compounds by the US Agency for Toxic Substances and Disease Registry (ATSDR, 2005), the toxicological review of zinc and compounds by the US Environmental Protection Agency (US-EPA (2005) Toxicological review of zinc and compounds. Washington, DC, USA) or the review by the WHO (WHO (2001). Environmental Health Criteria 221 Zinc). The results of the key experimental studies addressing potential effects of zinc compounds on fertility are summarised in the CSR.

Justification for selection of Effect on fertility via oral route:
Well documented two generation study, meets generally accepted scientific principles, acceptable for assessment.

Effects on developmental toxicity

Description of key information

Several prenatal toxicity studies are available that examined the developmental toxicity of various zinc compounds in rats, mice, rabbit or hamsters up to dietary exposure levels of 200 mg Zn/kg bw/day or 50 mg Zn/kg bw/day by gavage. No developmental toxicity has been observed in these studies and the NOAEL’s have been established at the highest doses tested.
Although some developmental effects such as decreases in body weights or decrease in individual organ weights were observed in F1 and/or F2 generations in the one or two generation reproductive toxicity studies conducted by Khan et al. (2007) at high exposure levels, these observations are, however, not suitable for risk assessment or hazard classifications as they were always accompanied with maternal toxicity. Moreover, no developmental toxicity was observed at non-maternally toxic doses in a teratogenicity study in which CF-1 albino mice were administered intraperitoneally 0, 12.5, 20.5 and 25 mg/kg on Day 11 of gestation (test 1) and at 20.5 mg/kg on Days 8 -11 of gestation (test 2) (Chang et al., 1979).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

50 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Additional information

The developmental toxicity of zinc compounds can be assessed on the basis of prenatal toxicity studies that have been conducted with soluble zinc sulphate and zinc chloride and slightly soluble zinc carbonate in rats, mice, hamsters or rabbits. Moreover, a total of three one or two generation reproductive toxicity studies conducted by Khan et al,.(Khan AT, Atkinson A, Graham TC, Green M, Ali S, Thompson SJ and Shireen KF (2001) Effects of low levels of zinc on reproductive performance of rats. Environ Sci 8: 367—381;

Khan AT, Atkinson A, Graham TC, Thompson SJ, Ali S and Shireen KF (2003) Effects of low levels of zinc on reproductive performance of mice. Environ Sci 10: 279—290;

Khan AT, Graham TC, Ogden L, Salwa AS, Thompson SJ, Shireen KF, and Mahboob M (2007). A two-generational reproductive toxicity study of zinc in rats. J. Environ. Sci. Health Part B 42: 403–415)

provide further information on potential teratogenic effects of zinc compounds.

No prenatal toxicity was observed with either zinc sulphate, zinc chloride or zinc carbonate at exposure levels up to 50 mg Zn/kg bw/day by oral gavage or 200 mg Zn/kg bw/day if the zinc was dose via the diet. Established NOAELs in these studies were typically at highest dose tested and systemically tolerated by the dams. Developmental effects such as decrease in body or organ weights were, however, observed in F1 and/or F2 generations in the one or two generation reproductive toxicity studies conducted by Khanet al. (Khan AT, Atkinson A, Graham TC, Green M, Ali S, Thompson SJ and Shireen KF (2001) Effects of low levels of zinc on reproductive performance of rats. Environ Sci 8: 367—381;

Khan AT, Atkinson A, Graham TC, Thompson SJ, Ali S and Shireen KF (2003) Effects of low levels of zinc on reproductive performance of mice. Environ Sci 10: 279—290;

Khan AT, Graham TC, Ogden L, Salwa AS, Thompson SJ, Shireen KF, and Mahboob M (2007). A two-generational reproductive toxicity study of zinc in rats. J. Environ. Sci. Health Part B 42: 403–415). These studies are not considered suitable for the assessment of teratogenic effects for hazard classification or risk assessment purposes since they were always observed in the presence of maternal toxicity.

In studies with women receiving zinc supplementation during pregnancies at levels of approximately ≤ 0.3 mg Zn/kg bw/day, no reproductive or developmental effects were observed ( WHO (2001). Environmental Health Criteria 221 Zinc; SCF (2003) Opinion of the Scientific Committee on Food on the tolerable upper intake level of zinc. Scientific Committee on Food SCF/CS/NUT/UPPLEV/62. Final, 19 March 2003 (expressed on 5 March 2003), Brüssel, Belgien). Evidence of zinc toxicity during human pregnancy has not been reported, but this may be due to the fact that very high exposures to zinc in human pregnancy are unusual. In contrast, zinc is necessary for normal growth and development (e.g., gene expression, vitamin metabolism) and therefore it is not surprising that zinc deficiency during pregnancy can cause a variety of adverse effects to the foetus or may result in reduced fertility or delayed sexual maturation in animals as well as in humans (EU RAR, 2008; WHO, 2001).

Overview of experimental studies on developmental toxicity

Test substance*	Species	Route	Method	Result	Remark	Reference
Zinc sulphate	Mouse CD-1	Oral	Females received daily doses of 0, 0.3, 1.4, 6.5 and 30 mg ZnSO4(unspecified)/kg bw by oral gavage during days 6-15 of gestation.	No discernible effects were seen on or maternal or foetal survival. No difference in number of abnormalities found in foetuses. NOAEL: 30 mg/kg bw/day equalling 12mg Zn/kg bw/d (anhydrate); 6.8mg Zn/kg bw/d (heptahydrate);	2 (reliable with restrictions) Key study	Food and Drug Research Labs., Inc (1973). Teratologic evaluation of FDA 71-49 (zinc sulfate). Report no.: PB-221 805. [Cited from EU 2004, a-f]. *
Zinc sulphate	Rat Wistar	Oral	Females received daily doses of 0, 0.4, 2.0, 9.1 and 42.5 mg ZnSO4(unspecified)/kg bw by oral gavage during days 6-15 of gestation.	No discernible effects were seen on or maternal or foetal survival. No difference in number of abnormalities found in foetuses. NOAEL: 30 mg/kg bw/day equalling 17mg Zn/kg bw/d (anhydrate); 9.6 mg Zn/kg bw/d (heptahydrate);	2 (reliable with restrictions) Key study	Food and Drug Research Labs., Inc (1973). Teratologic evaluation of FDA 71-49 (zinc sulfate). Report no.: PB-221 805. [Cited from EU 2004, a-f]. *
Zinc sulphate	Rat Charles Foster	Oral	Females received daily doses of 0, and 200 mg Zn/kg bw (in form of ZnSO4) in diet during days 1-18 of gestation	No discernible effects were seen on or maternal or foetal survival. A reduced number of implantations observed. No difference in number of abnormalities found in foetuses. NOAEL: 200 mg/kg bw/day	2 (reliable with restrictions) Key study	EU RAR, 2008
Zinc sulphate	Hamster	Oral	Females received daily doses of 0, 0.9, 4.1, 19, and 88 mg ZnSO4(unspecified)/kg bw by oral gavage during days 6-10 of gestation.	No discernible effects were seen on or maternal or foetal survival. No difference in number of abnormalities found in foetuses. NOAEL: 20 mg/kg bw/day	2 (reliable with restrictions) Key study	Food and Drug Research Labs., Inc (1973). Teratologic evaluation of FDA 71-49 (zinc sulfate). Report no.: PB-221 805. [Cited from EU 2004, a-f]. *
Zinc sulphate	Rabbit Dutch	Oral	Females received daily doses of 0, 0.6, 2.8, 13 and 60 mg ZnSO4(unspecified)/kg bw during days 6-18 of gestation.	No discernible effects were seen on or maternal or foetal survival. No difference in number of abnormalities found in foetuses. NOAEL: 13.6 mg/kg bw/day	2 (reliable with restrictions) Key study	Food and Drug Research Labs., Inc (1974b). Teratologic evaluation of compound FDA 71-49. Zinc sulfate in rabbits. Not reported. Testing laboratory: Food and Drug Research Labs., Inc. Report no.: PB-267 191. [Cited from EU 2004, a-f]. *
Zinc carbonate	Rat Sprague Dawley	Oral	Females received daily doses of 0, 2.5, and 50 mg Zn/kg bw (in form of ZnCO3) in diet during days 1-20 of gestation.	No discernible effects were seen on or maternal or foetal survival. No difference in number of abnormalities found in foetuses. NOAEL: 50 mg/kg bw/day	2 (reliable with restrictions) Key study	Uriu-Hare JY, Stern JS, Keen CL (1989) Influence of maternal dietary Zn intake on expression of diabetes-induced teratogenicity in rats Diabetes38(10):1282-90.

Toxicity to reproduction: other studies

Additional information

Effects in Fertility, Human information

In reviews by the World Health Organisation in the Environmental Health Criteria for Zinc ( WHO (2001). Environmental Health Criteria 221 Zinc) and by the US Agency for Toxic Substances and Disease Registry in the Toxicity Profile for Zinc ( ATSDR (Agency for Toxic Substances and Disease Registry) (2005). Toxicological profile for zinc. U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA, USA), existing human studies which examined the responses of women to zinc supplementation during pregnancy have been summarised. Studies on large controlled trials that were conducted to investigate the effects of dietary zinc supplementation in healthy pregnant women were peer reviewed. The reviewers concluded that zinc at a rate of 20mg/day and 30 mg/day did not result in any adverse reproductive effects during pregnancy ( Hunt IF, Murphy NJ, Cleaver AE, Faraji B, Swendseid ME, Coulson AH, Clark VA, Browdy BL, Cabalum MT, & Smith JCJ (1984). Zinc supplementation during pregnancy: effects on selected blood constituents and on program and outcome of pregnancy in low income women of Mexican descent.Am J Clin Nutr, 40: 508–521; Kynast G and Saling E (1986) Effect of oral zinc application during pregnancy. Gynecol Obstet Invest, 21: 117–123).Two exemplar studies are summarised in the following:

A double blind trial was conducted in 56 pregnant women at risk of delivering a small for gestational-age baby to determine the effects of dietary zinc supplementation during the last 15-25 weeks of pregnancy following administration of 22.5 mg zinc/day. No adverse reproductive effects were observed ( Simmer K, Lort-Phillips L, James C, et al. 1991.A double-blind trial of zinc supplementation in pregnancy. Eur J Clin Nutr 45:139-144).

Pregnant women who received 0.3 mg zinc/kg/day as zinc sulphate capsules during the last two trimesters did not exhibit any changes in maternal body weight gain, blood pressure, postpartum haemorrhage or infection, inidicating no adverse reproductive effects ( Mahomed K, James DK, Golding J and McCabe R (1989). Zinc supplementation during pregnancy: A double blind randomised controlled trial. Br. Med. J. 299: 826-30).

Developmental toxicity Human information

In establishing the Environmental Health Criteria for Zinc, the World Health Organisation has reviewed and summarised existing human studies examining the responses of women to zinc supplementation during pregnancy. None of the studies indicated any significant effects on the developing foetus ( WHO (2001). Environmental Health Criteria 221 Zinc). Two exemplar studies are summarised in the following:

A study was conducted on pregnant women to determine the effects of nutrients during pregnancy on maternal and fetal outcome. Four hundred fifty women were observed during pregnancy and postpartum. Forty-three variables including 12 laboratory indices of maternal nutrient status were assessed. Maternal plasma zinc levels were inversely correlated with fetal weight. Blood examinations revealed a significant association between the total occurrence of fetomaternal complications or fetal distress, and lowest quartile zinc/albumin and highest quartile folate. Under the study conditions, plasma zinc was determined to be a discriminator for fetomaternal complications only in women in the lowest quartile for plasma zinc ( Mukherjee MD, Sandstead HH, Ratnaparkhi MV, Johnson LK, Milne DB and Stelling HP (1984). Maternal zinc, iron, folic acid, and protein nutriture and outcome of human pregnancy. Am. J. Clin. Nutr. 40(3):496-507).

A double blind trial was conducted on pregnant women to determine the effects zinc supplementation during pregnancy on maternal and fetal outcome. 494 women booking before 20 week of gestation in a hospital were prescribed either 66 mg zinc sulphate (equivalent to 20 mg elemental zinc) capsules or placebo for once daily use, starting from day of booking till delivery. Various adverse outcomes were tested, including maternal bleeding, hypertension, complications of labour and delivery, gestational age, Apgar scores, and neonatal abnormalities. The main outcome measure was birth weight. There were no differences between the mothers and neonates of the zinc supplemented and placebo group. Under the test conditions, zinc supplementation during pregnancy did not affect maternal or fetal outcome ( Mahomed K, James DK, Golding J and McCabe R (1989). Zinc supplementation during pregnancy: A double blind randomised controlled trial. Br. Med. J. 299: 826-30).

Justification for classification or non-classification

There is no experimental evidence that would justify a classification of zinc compounds for hazardous effects for reproductive or developmental toxicity according under the Dangerous Substance Directive 67/548/EEC or Regulation (EC) 1272-2008 on the classification, labelling and packaging of substances and mixtures. The available reproductive and developmental toxicity information has been exclusively generated with soluble zinc compounds zinc chloride or zinc sulphate which ensure maximum bioavailable concentration of zinc and hence, allow the use of the information also for the assessment of the slightly soluble zinc compounds and insoluble zinc metal on a read across basis. No experimental fertility data were identified for these compounds.