Naphthenic acids, zinc salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no data for zinc naphthenate. Data from appropriate read across substances are presented, with data on zinc compounds and naphthenic acids.

 

There were no treatment-related effects on any of the assessed reproductive parameters in an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study involving the exposure of rats to a mixture of naphthenic acids by oral gavage at up to 900 mg/kg bw/day. The reproductive NOAEL was therefore established as 900 mg/kg bw/day (HPVIS, 2010).

 

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, 2004), 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) or the review by the WHO (WHO, 2001). The NOAEL was determined to be 20 mg zinc/kg bw/day.

 

The sample of zinc naphthenate used for hazard testing (taken as representative of the range of potential compositions of zinc naphthenate) contains approximately 14.3% zinc, therefore the NOAEL value has been recalculated for zinc naphthenate. The NOAEL for zinc naphthenate would be 137.9 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, key study used in EU risk assessment report for Zinc metal

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

according to 408 in which reproductive paremeters were also observed.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

see section 7.5.1 Repeated dose toxicity, 90 d, rat, oral, zinc monoglycerolate, Edwards & Buckley et al, 1995, Sup, KL2

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

section 7.5.1 Repeated dose toxicity, 90 d, rat, oral, zinc monoglycerolate, Edwards & Buckley et al, 1995, Sup, KL2

Details on mating procedure:

none

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

section 7.5.1 Repeated dose toxicity, 90 d, rat, oral, zinc monoglycerolate, Edwards & Buckley et al, 1995, Sup, KL2

Frequency of treatment:

section 7.5.1 Repeated dose toxicity, 90 d, rat, oral, zinc monoglycerolate, Edwards & Buckley et al, 1995, Sup, KL2

Details on study schedule:

section 7.5.1 Repeated dose toxicity, 90 d, rat, oral, zinc monoglycerolate, Edwards & Buckley et al, 1995, Sup, KL2

Remarks:

Doses / Concentrations:

Basis:

No. of animals per sex per dose:

20 animals per sex

Control animals:

yes, plain diet

Details on study design:

section 7.5.1 Repeated dose toxicity, 90 d, rat, oral, zinc monoglycerolate, Edwards & Buckley et al, 1995, Sup, KL2

Dose descriptor:

NOAEL

Effect level:

60 other: mg Zn2+/kg bw/day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Conclusions:

Male and female rats were exposed to zinc monoglycerolate up to 1% in the diet, equal to ca.335 mg Zn2+/kg bw/day for 58 days, after which the concentration in the feed was decreased for one week to 0.5%, equal to ca.300 mg Zn2+/kg bw/day. Subsequently, the animals had to be killed at day 64 because of poor health and compromised food consumption (note also the non-linearity in the Zn2+-doses). The testes of all these males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female the uterus was hypoplastic. All other rats exposed to 0.05 or 0.2% (ca.13 or 60 mg Zn2+/kg bw/day, respectively) survived to the end of the 13 weeks treatment, without showing detrimental effects on sex organs.

Executive summary:

In the repeated dose toxicity 90 day study in rats fed zinc monoglycerolate up to 1% in the diet, equal to ca.335 mg Zn²⁺/kg bw/day for 58 days, after which the concentration in the feed was decreased for one week to 0.5%, equal to ca.300 mg Zn²⁺/kg bw/day. Subsequently, the animals had to be killed at day 64 because of poor health and compromised food consumption (note also the non-linearity in the Zn²⁺-doses). The testes of all these males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female the uterus was hypoplastic. All other rats exposed to 0.05 or 0.2% (ca.13 or 60 mg Zn²⁺/kg bw/day, respectively) survived to the end of the 13 weeks treatment, without showing detrimental effects on sex organs.

Reference 2

Endpoint:

toxicity to reproduction

Remarks:

other: combined repeated dose & reproductive/developmental toxicity screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed by NTP according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classification. The score 1 was given bij HPVIS.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The naphthenic acids were suspended in corn oil to the appropriate concentrations and administered in 10 ml/kg doses.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days. All females confirmed to have mated were placed in plastic maternity cages once mating was confirmed.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Females for which copulation was not detected were placed in maternity cages at the end of the 1
- Any other deviations from standard protocol:Length of gestation was calculated as the time from confirmation of mating to the onset of delivery.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Dosing of males was initiated 14 days prior to pairing and throughout a 14 day mating period for a total of 28-29 doses. Dosing of females was also initiated 14 days prior to pairing and continued throughout the mating and gestational periods until study termination on post-natal day 3. The total number of doses ranged from 39-53 depending on the time at which mating occurred.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 100, 300, 900 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- All rats were examined twice daily for mortality and general health.
- All animals were examined approximately 1 hour after each treatment, and all unusual observations were recorded.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:Detailed physical examinations of all animals were conducted weekly (See Section 7.5.1)

BODY WEIGHT: Yes
- Time schedule for examinations: females: recorded once week prior to test substance administration, on the first day of dose administration and weekly until evidence of copulation was obtained. From that point body weights of female rats were recorded on gestation days (GD) 0, 4, 7, 11, 14, 17, and 20 and on lactation days (LD) 0, 1 and 4 (termination). For females for which there was no evidence of copulation, body weights were recorded weekly until termination.

FOOD CONSUMPTION:
- Food consumption by adult animals was also recorded on the same schedule as the body weights.

WATER CONSUMPTION: No

OTHER:
- Parental mating, fertility, conception and copulation indices , gestation length, numbers of former implantation sites, absolute and relative organ weights, and pre-coital intervals.
- Toxicological parameters: See Section 7.5.1.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
On the day of parturition, all pups were examined for viability, for the presence of gross malformations and to assess gender. The numbers of live and stillborn pups were recorded.
All offspring were uniquely identified and examined daily for signs of mortality and ill health. All offspring were individually weighed on PND 1 and 4. Gender was assessed on PND 0 and 4. At scheduled termination, PND 4, all surviving offspring were euthanized and discarded without further examination.

GROSS EXAMINATION OF DEAD PUPS: no data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: 14 days after mating
- Maternal animals: . Females for which there was no evidence of mating were sacrificed on post-cohabitation day 25, those that showed evidence of mating but failed to deliver were euthanized on post-mating day 25, and all others were euthanized on post-natal day 4.

GROSS NECROPSY: YES
- At termination rats were euthanized by carbon dioxide inhalation.
- Necropsies were conducted on all animals sacrificed in extremis or at study termination.

HISTOPATHOLOGY / ORGAN WEIGHTS
- An examination of target organs including male and female reproductive organs was also carried out as part of this test (See also Section 7.5.1).
- Organs examined included: ovaries with oviduct, uterus with cervix and vagina, testes with epididymides, prostate and seminal vesicles.
- The ovaries, testes and uteri were weighed and all were examined histologically

Postmortem examinations (offspring):

All offspring were individually weighed on PND 1 and 4.
Gender was assessed on PND 0 and 4At scheduled termination, PND 4, all surviving offspring were euthanized and discarded without further examination.

Statistics:

Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates’ correction (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, numbers of corpora lutea, number of pups born, live litter size on PND 0, unaccounted for sites, absolute and relative organ weights, and pre-coital intervals were evaluated by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.

Reproductive indices:

See Tables below and in Section 7.8.2.
Mating, fertility, pregnancy and gestation indices were not provided as such, however No. of females mated, pregnant and with litters were given.
Pre-implantation loss not provided, but No. of corpora lutea and No. of implantation sites given.
Implanation index not provided, but No. of implanatation given.
Post-implantation indexes given.

Offspring viability indices:

Viability index not provided, but No. born and alive on day 4 given.
Sex ratio given (See Section 7.8.2)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

The absolute epididymal weights were increased in the 900 mg/kg/day group but were not significantly different when expressed on a per body weight basis. The uterine weights were also significantly elevated but this was considered to have been a consequence of the fact that the females were all in lactational anaestrous. The uterine weights were within the historical range of the laboratory and were not considered to have been toxicologically important. There were no weight differences in any of the other organs and no pathological changes in any of the reproductive organs at the highest dose tested (900 mg/kg/day).

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: mating index

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: reproductive organ effects

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Decrease No. born pups at 900 mg/kg bw - See Section 7.8.2

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decrease at 900 mg/kg bw - See Section 7.8.2

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: mating index

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: reprodiuctive organ effects

Reproductive effects observed:

not specified

Table 1. Summary of reproductive parameters assessed in the repeated dose/reproductive toxicity study of refined naphthenic acids.

Dose (mg/kg/day)	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Number of females paired	12	12	12	12
Number of female mated	12	12	10	11
Number of females pregnant^a	9	12	10	11
Number of females with litters	9	12	10	11
Pre-coital interval (days)^b	1.4+0.7	2.3+1.1	4.2+3.3^c	3.8+3.5
Gestation length (days)	21.4+0.6	21.9+0.3	22.0+0.5	22.1+0.5
Corpora lutea	15.6+2.3	14.0+1.4	15.1+3.0	13.8+2.1
Implantation sites	15.0+2.4	13.6+1.1	13.0+1.2	12.2+3.7
Number born	14.1+1.9	12.9+1.1	12.0+1.6	10.8+3.8^c
Post-Implantation loss (%)^d	6.0	5.1	7.7	11.5

a. Pregnant = uterine implantation sites.

b. Data summarized as mean+standard deviation.

c. p < 0.05

d. Post-implantatoin loss = (No. of implantations - No. of life fetuses)/No. of implanatations (%)

Conclusions:

No reproductive effects were identified. The NOAEL for mating and reproductive effects of is 900 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar ratswas performed by oral gavage with Naphtenic acids in corn oil. There were 3 test material treated groups (100, 300 and 900 mg/kg bw) along with a vehicle treated group (corn oil) each in 12 animals/sex/group. Male rats were dosed during premating, mating and afterwards for 28 days in total and females were dosed during premating, mating, gestation and up to day 3 post partum. In this section, only reproductive toxicity parameters are discussed: (furher info on repeated & developmental parameters is given in Section 7.5.1 and 7.8.2. Target organ findings were identified at the dose of 900 mg/kg bw, whereas 100 mg/kg bw was considered as the NOAEL for systemic toxicity.

No reproductive effects were identified up to 900 mg/kg bw . There were no weight differences in any of the other organs nor any pathological changes in the reproductive organs up to the highest dose tested (900 mg/kg/day). The NOAEL for mating and reproductive organ effects was 900 mg/kg/day.

Reference 3

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

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.

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes


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

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

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


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


GROSS EXAMINATION OF DEAD PUPS:
No

Postmortem examinations (parental animals):

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


HISTOPATHOLOGY / ORGAN WEIGHTS:

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

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

Postmortem examinations (offspring):

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

Statistics:

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

Reproductive indices:

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

Offspring viability indices:

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

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

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


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


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


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

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


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


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


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

Dose descriptor:

LOAEL

Effect level:

7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

haematology

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

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


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


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


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


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


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

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


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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

None

Conclusions:

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

Executive summary:

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

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

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

Reference 4

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting 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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Males rats were fed diet supplemented with the test material for specific period and then mated with normal females. Males were sacrificed after mating and effect on sperm motility/viability and zinc concentration in reproductive organs observed. Females were allowed to have full term gestation and effect on conception and litter viability were determined.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Charles-Foster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: (P) Males: 162 g
- Diet: Crushed rat feed of Hindustan Lever (India); ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Crushed rat feed

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1 d
- Proof of pregnancy: Sperm in vaginal smear (mating performed only once irrespective of results)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

30-32 d before mating to males only

Frequency of treatment:

Daily, ad libitum

Details on study schedule:

Following the day of mating, males were sacrificed and sperm was immediately collected from epididymis for motility and viability studies. Reproductive organs were dissected out for estimation of zinc. Mated females were allowed to have full term gestation.

Remarks:

Doses / Concentrations:
4,000 ppm zinc as zinc sulphate
Basis:
nominal in diet

No. of animals per sex per dose:

Control group: 15
Treatment group: 18

Control animals:

yes, plain diet

Details on study design:

None

Positive control:

None

Parental animals: Observations and examinations:

No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in male parents:
Sperm motility and viability

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number of pups, stillbirths, live births and malformation

Postmortem examinations (parental animals):

- Male animals: All animals sacrificed following the day of mating and reproductive organs (testis, epididymis, seminal vesicle and prostate) were dissected out for estimation of zinc
- Maternal animals: Not sacrificed

Postmortem examinations (offspring):

No data

Statistics:

Fischer's one sided T test

Reproductive indices:

None

Offspring viability indices:

None

Clinical signs:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Motility of the sperm was significantly reduced in the zinc treated rats at all time intervals viz. 30 min., 2 h and 4 h. The percentage reduction as compared to the controls was 8.5, 25 .3 and 29 .0 respectively. Sperm viability was unaffected. (See Table 3 for details)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Mating by zinc treated males caused significant lowering of incidence of conception and number of live births per mated female. Only 11 females conceived out of 18 mated by zinc treated males whereas all females mated with control rats conceived. (See Table 1 for details)

OTHER FINDINGS (PARENTAL ANIMALS): Zinc content was significantly increased only in the testis (25 %) and sperm (18 %) of the zinc treated rats. (See Table 2 for details)

Dose descriptor:

dose level: 4,000 ppm zinc

Sex:

male

Basis for effect level:

other: overall effects sperm characterization; other: zinc concentration in reproductive organs

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

other: Not treated but mated with males fed 4,000 ppm zinc

Sex:

female

Basis for effect level:

other: Overall effects incidence of conception

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

VIABILITY (OFFSPRING): No stillbirth in any of the groups

OTHER FINDINGS (OFFSPRING): No malformed litter in any of the groups

Dose descriptor:

other: Offsprings of 4,000 ppm zinc treated males

Generation:

F1

Sex:

male/female

Basis for effect level:

other: overall effects live births; offspring malformation

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Reproductive effects observed:

not specified

Table 1: Outcome of mating by zinc treated male rats

	Mating males
	Control	Experimental
Number of females mated	15	18
Number of females conceived	15	11
Normal live litter
Total number	101	61
Number / mated female	6.73	3.38*

*Difference was significant at 5% level

Table 2: Zinc content of the reproductive tissues of zinc treated male rats

	Control	Experimental
Testis	28.1± 1.63 (15)	35.3 ± 2.13 (18)*
Epididymis	58.3 ± 3.46 (10)	61.4 ± 6.63 (10)
Seminal vesicle	24.4 ± 3.25 (10	33.8 ± 5.96 (10)
Prostate (whole)	48.8 ± 6.90 (10)	51.7 ± 6.36 (10)
Sperm	559 ± 14.8 (10)	658 ± 24.7 (10)*

Results expressed as Mean ± SEM in µg/g, except for sperm in µg/g dw, for the number of

rats shown in parentheses.

* P < 0.02

Table 3: Motility and viability of the sperm of zinc treated rats

	Control (15)	Experimental (18)
Motility (Emmen's unit)
30 min	25.9 ± 0.57	23.7 ± 0.47*
2 hours	23.7 ± 0.58	17.7 ± 1.15**
4 hours	17.2 ± 1.10	12.2 ± 1.42*
% viable at 4 hours	55.7 ± 4.80	49.0 ± 4.60

Values expressed as Mean ± SEM for the number of rats shown in parentheses.

* P < 0.01 

**P < 0.001

Conclusions:

Dietary zinc supplementation at 4,000 ppm reduced male fertility in rats under the conditions of the study.

Executive summary:

A study 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 d. 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

137.9 mg/kg bw/day

Species:

rat

Additional information

There are no data for zinc naphthenate. Data from appropriate read across substances are presented, with data on zinc compounds and naphthenic acids.

 

Naphthenic acids

In a combined repeated dose toxicity and reproduction/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, Sprague-Dawley rats (12/sex/group) were orally administered a mixture of naphthenic acids by stomach tube (gavage, in corn oil) at doses of 0, 100, 300 or 900 mg/kg bw/day. Males were dosed for 28 days (during the pre-mating, mating and post-mating periods) and females were dosed through pre-mating, mating, gestation and up to day 3 post partum (39-53 days in total).There were no test item-related adverse effects on the measured reproductive parameters (including mating index, precoital time, gestation length, corpora lutea and post-implantation loss) nor any microscopic effects on the reproductive organs. Consequently, the NOAEL for reproductive toxicity was set at 900 mg/kg bw/day, the highest dose tested (HPVIS, 2010). [See Repeated dose toxicity and Effects on developmental toxicity sections for further details].

 

In a non-guideline study, a group of 12 male New Zealand white rabbits received dermal applications of 2 ml undiluted calcium naphthenate (in an unspecified ‘carrier oil’) for 6 hours/day on 5 days/week for 10 weeks. A control group of 12 male rabbits was similarly exposed to the vehicle only. After the exposure period each male was mated with 2 untreated females. Males were necropsied either directly after mating or approximately 12 weeks later; gross and microscopic examinations of the male reproductive tracts were conducted. The females were necropsied on day 29 of gestation. Numbers of corpora lutea, total implantations, pre-and post-implantation losses and numbers of viable foetuses were recorded. Calcium naphthenate (in ‘carrier oil’) showed no effects on any of the evaluated parameters (Dix and Cassidy, 1983).

 

In a non-guideline study, female Wistar rats (13-14/group) were gavaged with a mixture of naphthenic acids (isolated from Athabasca oil sands, in corn oil) at doses of 0, 6 or 60 mg/kg bw/day for a total of 39-53 days (through 14 days pre-mating, mating, gestation and up to post-natal day 3). A dramatic effect on female fertility was observed in the high-dose group, with only a single female successfully bearing a litter; total cholesterol was significantly reduced at this dose. Mating and ovulation were unaffected by treatment (Rogers, 2003). These results suggest that the dose-related female infertility may be associated with poor embryonic implantation, an effect that might be secondary to depressed sex hormone production requiring cholesterol as a precursor. [See Effects on developmental toxicity section for further details].

 

Zinc

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 et al.,2001, 2003, 2007; Samanta et al, 1986). 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.(1981) and Edwards and Buckley (1995).

 

Maita et al. (1981) reported that mice and rats fed with zinc sulphate in dietary concentrations up to 30,000 mg/kg feed did not produce adverse effects on either male or female sex organs after 13 weeks of exposure. This dietary level was equal to ca. 1100 mg or 565 mg Zn/kg bw/day for mice and rats, respectively. Edwards and Buckley (1995) showed that 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. In the exposure group of 335 mg Zn/kg bw/day, all males showed hypoplasia in testes and seminiferous tubules in males hypoplastic uterus in females, but these findings are not considered reliable as the animals of this high dose group were generally of poor health conditions and killed for humane reasons prior to study termination.

 

In addition to those key reproductive toxicity studies (Khan et al.,2001, 2003, 2007; Samanta et al, 1986), some additional studies indicating high oral doses of zinc (i.e. exposures greater that 25 mg day/kg bw/day) to impair fertility as indicated by a decreased number of implantations sites and increased number of resorptions are of note (EU RAR, 2004; Pal et al., 1987).

 

A study was carried out 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 (Kumar et al., 1976). The low protein diet may have affected the physiology of the animals resulting in an increased sensitivity for zinc. As this hypothesis cannot be further assessed and also considering the limited available study information, this study is only of limited validity for the assessment of effects of zinc exposure on fertility (EU RAR, 2004).

 

Another study aimed at determining 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 (i.e., approximately 200 mg Zn/kg bw/day) 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 days pre-mating period, maximum 5 days of mating period and 18 days of post-coitum period. All the females were sacrificed on Day 18 of gestation and uterus content and fetuses 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 fetal and placental weights, stillbirths and malformed fetuses 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 fetal 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 (Pal et al., 1987).

 

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 study non-zinc relevant study specificities could have impacted the study outcome.

 

Furthermore, in reviews by the World Health Organisation in the Environmental Health Criteria for Zinc (WHO, 2001) and by the US Agency for Toxic Substances and Disease Registry in the Toxicity Profile for Zinc (ATSDR, 2005), 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 20 mg/day and 30 mg/day did not result in any adverse reproductive effects during pregnancy (Hunt et al.,1984; Kynast and Saling et al.,1986).

 

Two exemplar studies are summarised are Simmer et al (1991) and Mahomed et al (1989). Simmer et al (1991) conducted a double blind trial 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. In Mahomed et al (1989), pregnant women who received 0.3 mg zinc/kgbw/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, indicating no adverse reproductive effects.

Effects on developmental toxicity

Description of key information

There are no data for zinc naphthenate. Data from appropriate read across substances are presented, with data on zinc compounds and naphthenic acids.

 

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study, a mixture of naphthenic acids was administered to rats by oral gavage at doses of 0, 100, 300 or 900 mg/kg bw/day. The developmental NOAEL was considered to be 100 mg/kg bw/day based on reductions in the number of offspring, live pups born, and offspring body weights observed at higher doses (HPVIS, 2010).

 

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 therefore NOAELs 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). The NOAEL was determined to be 50 mg zinc/kg bw/day.

 

The sample of zinc naphthenate used for hazard testing (taken as representative of the range of potential compositions of zinc naphthenate) contains approximately 14.3% zinc, therefore the NOAEL value has been recalculated for zinc naphthenate. The NOAEL for zinc naphthenate would be 344.8 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

344.8 mg/kg bw/day

Species:

rat

Additional information

There are no data for zinc naphthenate. Data from appropriate read across substances are presented, with data on zinc compounds and naphthenic acids.

 

Naphthenic acids

 

In a combined repeated dose toxicity and reproduction/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, Sprague-Dawley rats (12/sex/group) were orally administered a mixture of naphthenic acids by stomach tube (gavage, in corn oil) at doses of 0, 100, 300 or 900 mg/kg bw/day. Males were dosed for 28 days (during the pre-mating, mating and post-mating periods) and females were dosed through pre-mating, mating, gestation and up to day 3 post partum (39-53 days in total). A significant reduction in the number of live born pups was observed in the mid- and high-dose groups. The number of offspring and offspring body weights were also significantly reduced at the highest dose. Treatment with naphthenic acids did not produce malformations. The developmental NOAEL was considered to be 100 mg/kg/day (HPVIS, 2010). [See Repeated dose toxicity and Effects on fertility sections for further details].

 

In a non-guideline study, female Wistar rats (13-14/group) were gavaged with a mixture of naphthenic acids (isolated from Athabasca oil sands, in corn oil) at doses of 0, 6 or 60 mg/kg bw/day for a total of 39-53 days (through pre-mating, mating, gestation and up to post natal day 3). No malformations were apparent in the offspring (Rogers, 2003). [See Effects on fertility section for further details].

 

Zinc

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.(2001, 2003, 2007) provide further information on potential teratogenic effects of zinc compounds.

 

Several prenatal toxicity studies are available that examined the developmental toxicity of various zinc compounds 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 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. Established NOAELs in these studies were typically at highest dose tested and systemically tolerated by the dams.

 

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).

 

Furthermore, 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). Two exemplar studies are summarised in Mukherjee et al (1984) and Mahomed et al (1989).

 

A study was conducted on pregnant women to determine the effects of nutrients during pregnancy on maternal and foetal 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 foetal weight. Blood examinations revealed a significant association between the total occurrence of foetal-maternal complications or foetal distress, and lowest quartile zinc/albumin and highest quartile folate. Under the study conditions, plasma zinc was determined to be a discriminator for foetal-maternal complications only in women in the lowest quartile for plasma zinc (Mukherjee et al., 1984).

 

A double blind trial was conducted on pregnant women to determine the effects zinc supplementation during pregnancy on maternal and foetal 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, 0.3 mg zinc/kg bw/day) 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 foetal outcome (Mahomed et al.,1989).

 

In summary, 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; SCF, 2003). 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, 2004; WHO, 2001).

Justification for classification or non-classification

Not classified. Available data suggest no classification for reproductive and developmental toxicity.

 

There are no data for zinc naphthenate. Data from appropriate read across substances are presented, with data on zinc compounds and naphthenic acids. The NOAEL have been recalculated from zinc ions to zinc naphthenate. 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 mainly 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.