Zinc methacrylate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Zinc chloride

EC Number:

231-592-0

EC Name:

Zinc chloride

Cas Number:

7646-85-7

IUPAC Name:

zinc dichloride

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Zinc chloride
- Source: Fisher Scientific
- Analytical purity: > 97 %

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

ENVIRONMENTAL CONDITIONS
- Temperature: 70-78 °F
- Humidity: 50-55 %
- Air changes: 1/10 min
- Photoperiod : 12 h light/12 h dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

Details on mating procedure:

- Length of cohabitation: 21 days
- Proof of pregnancy: Conception (Day 0 of gestation) was checked daily in the mornings by looking for the presence or absence of copulatory plugs.
- After the cohabitation period, the mated females were separated from the males.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

2 generations: 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.

Frequency of treatment:

Once daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
7.5, 15, 30 mg/kg bw/day
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 Zinc chloride was set at 60 mg/kg bw/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 mg/kg bw/day) of the established MTD. Likewise, the mid-dose group (group 3) was at 1/4 (15 mg/kg bw/day) of the established MTD and the lowest dose group (group 2) was 1/8 (7.50 mg/kg bw/day) of the established MTD.

Positive control:

No data

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT AND FOOD CONSUMPTION: Yes
- Food efficiency = (body weight gain/amount of diet consumed) × 100

OTHER:
HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy, the F0 males and at Day 21 of lactation, all F0 females and one male and one female F1 offspring were anesthetized with a combination of intraperitoneal Pentothal and Isoflo via inhalation. Blood samples for hematology and clinical chemistries were collected in heparinised 3mL syringes via cardiac puncture.
- Blood Chemistry parameters: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, amylase, blood urea nitrogen, creatinine, cholesterol, sodium, potassium, chloride, calcium, phosphorus, albumin, total protein, total bilirubin, and glucose 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 (4 sex/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.
- Body weights 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

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 F0 parents.

GROSS EXAMINATION OF DEAD PUPS: No

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.
- When the ANOVA revealed significant differences among the treatment groups, Dunnett's and/or Duncan's multiple comparison procedures were utilized to pinpoint the specific treatment differences. Differences were considered statistically significant if p≤ 0.05.

Reproductive indices:

- Fertility index (%) = (number of females delivering/number of females cohabited) × 100

Offspring viability indices:

- Live birth index (%) = (number of live pups at Day 0/number of pups born) × 100
- 4-day survival index (%) = (number of live pups on Day 4/number of pups alive on Day 0) × 100
- 21-day (weaning) survival index (%) = (number of pups alive on Day 21/number of pups alive on Day 4) × 100
- Litter Size = Number of pups/number of pregnant females

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- Aggression/hyperactivity throughout the study in both F0 males and females, hair loss behind the ears in males, vaginal discharges in low and high dose females; 0, 8, 20 and 12 % mortality in males were observed in control, low, mid and high dose groups, respectively and 12, 24, 28 and 24 % mortality in females were observed in control, low, mid and high dose groups, respectively.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- All treated F0 males experienced significant reduction in body weight after the 1st week of dosing and this trend continued up to the end of the experiment. The total weight gain of males was significantly reduced (dose dependent) in the low-, mid- and high-dose groups. In the F0 females, total weight gain and percent reduction in the low- mid- and high-dose groups were not significantly different from the control.
- Zinc chloride treatment to F0 and F1 males and females caused no significant effects on their feed efficiencies when compared to their control groups.

HEMATOLOGY AND CLINICAL CHEMISTRY:
- None of the hemogram or leukogram values of both F0 and F1 males and females among the 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 Amylase, Alkaline phosphatase and glucose.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
- In F0 rats, Zinc chloride 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 mid and high-dose groups, the liver and kidney in all 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 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 Zinc chloride 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 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 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 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 treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the 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.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

other: not reported

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING):
- The F1 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 treated groups.

BODY WEIGHT (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 F1 females in the low-dose group experienced significant reductions in body-weight gain on weeks 15, 16, and F1 females in the mid-dose group experienced a similar reduction in body-weight gain weeks 3 through 17, This was also true for females in the highest dose group for weeks 1 through 17. 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 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 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 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 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 treated females generally paralleled those observed in their male counterparts.

HISTOPATHOLOGY (OFFSPRING):
- In F1 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) at 30 mg/kg bw/day. These results indicated that Zinc chloride 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 treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the 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, Zinc chloride treatment resulted in a significant reduction on fertility, viability (Day 0) and litter size in the high-dose group compared to control. However, the treatment showed no effect on viability index, weaning index and sex ratios of F2 pups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of Zinc chloride was 7.5 mg/kg bw/day in rats.

Executive summary:

In a two-generation study conducted similarly to OECD Guideline 416, Zinc chloride was administered to groups of Sprague-Dawley rats (25/sex/dose) at 7.5, 15 and 30 mg/kg bw/day by oral gavage. Treatment was started after two weeks of acclimation and was continued for males and females for 77 days prior to cohabitation. Treatment was continued throughout the periods of cohabitation (21 days) for both sexes. Treatment of female rats was continued throughout the gestation (21 days) and lactation (21 days) periods. Control group animals received deionised water. During the study, data was recorded on mortality, clinical signs, body weight change, food consumption, haematology, blood chemistry, and reproductive performance. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken. 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. Aggression/hyperactivity was observed throughout the study in both F0 and F1 males and females of treated groups. Exposure of Zinc chloride to F0 and F1 parental males resulted in a significant reduction in their body weights, and the F0 and F1 parental females did not show any significant difference in their body weights compared to their control groups. However, the postpartum dam weights of both F0 and F1 female rats were significantly reduced compared to their controls. Zinc chloride treatment to F0 and F1 males and females caused no significant effects on their feed efficiencies when compared to their control groups. Exposure of test material to F0 and F1 generation parental animals resulted in non significant change in clinical pathology parameters, except the alkaline phosphatase level, which showed an upward trend in both sexes of both generations. In F0 rats, reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females were observed. In F1 rats, reduction of brain, liver, kidney, adrenal, spleen, prostate and seminal vesicles weights of males and in spleen and uterus of females were observed. Gross lesions were observed in gastro-intestinal (GI) tract, lymphoreticular/ hematopoietic and reproductive tract in parental rats in both generations. 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 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 treated females generally paralleled those observed in their male counterparts. In both F0 and F1 males, the histopathological 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 treated groups. No significant changes in clinical pathology values or organ weights correlated with these lesions and none of the microscopic changes in target organs were of great magnitude. All unscheduled deaths were confined to the treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the treated females was similar to that seen in the males, except that no lesions were seen in the reproductive system. Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of Zinc chloride was 7.5 mg/kg bw/day in rats (Khan, 2007).