mancozeb (ISO); manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt  

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-06-14 to 2015-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Complexation products of manganese and zinc with ethylenebis(dithiocarbamate)

EC Number:

616-995-5

Cas Number:

8018-01-7

Molecular formula:

(x+y)[C4H6N2S4]2- + xMn2+ + yZn2+, x:y ranges between 1:0.062 to 1:0.12 (mean 1:0.091)

IUPAC Name:

Complexation products of manganese and zinc with ethylenebis(dithiocarbamate)

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.,
- Age at study initiation: approx. 93 days
- Weight at study initiation: not specified
- Fasting period before study: no
- Housing: individually, in clean, stainless steel wire-mesh cages suspended above cage-board
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 43.3 - 66.1 %
- Air changes: 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1.0% (w/v) methylcellulose in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle suspension was prepared approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated, protected from light. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle: well stability of the test item and recommended by guidelines
- Concentration in vehicle: 0, 1, 4, 16 mg/mL
- Amount of vehicle: 10 mL/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All dose formulation samples were analyzed for test substance concentration using a method previously validated by the WIL Research Analytical Chemistry Department.

Test substance homogeneity was assessed and verified in formulations prepared at concentrations of 0.1 and 50 mg mancozeb/mL in different batch sizes (100 and 700 mL). Following at least 10 days of room temperature storage or refrigerated storage, resuspension homogeneity was assessed and verified in formulations prepared at concentrations of 0.1 and 50 mg mancozeb/mL stored at 2 different aliquot sizes (25 and 100 mL). Finally, test substance stability was assessed and verified in formulations prepared at target concentrations of 0.1 and 50 mg mancozeb/mL and stored at room temperature for at least 18 hours or refrigerated for 11 days. Therefore, stability and resuspension homogeneity analyses were not conducted on this study.
Samples for concentration analysis were collected from the middle stratum of the first and last dosing formulation (including the control group). One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored refrigerated (2°C to 8°C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

14 days (GD 6 - 19 )

Frequency of treatment:

daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

vehicle control

No. of animals per sex per dose:

25 / pregnant females / dose

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale:
Dosage levels were selected based on a range-finding prenatal developmental toxicity study with mancozeb at dosage levels of 80, 120, and 160 mg/kg/day that was preceded by a tolerability study of mancozeb in non-pregnant rats at 60, 120, 180, 240, and 300 mg/kg/day. In the range-finding prenatal developmental toxicity study, mean maternal body weight gain and food consumption were lower throughout the gestation dosing period (gestation days 6-20) in the 160 mg/kg/day mancozeb group and resulted in an approximately 21.6% decrease in mean body weight gain during gestation days 6-20. A high dosage level of 160 mg/kg/day mancozeb was therefore chosen as the highest dosage level that elicits maternal toxicity in the current study. The lower dosage levels were chosen to assess the dose-response relationship. The primary metabolite of mancozeb, ETU, is a known rat teratogen, and ETU-related developmental toxicity (fetal malformations) was observed at 15 and 30 mg/kg/day in a prenatal developmental toxicity study with ETU (Edwards, 2015, WIL-83504). However, there was no significant maternal toxicity noted in the ETU study at any dosage level. The highest dosage level in the present study was therefore based upon the maternal tolerability of mancozeb. Toxicokinetic measurements made in the previous studies have demonstrated that the fetus is exposed to both mancozeb and ETU following maternal dosing, with an ETU maternal:fetal ratio of approximately 1.0. The selected route of administration for this study was oral (gavage) because this is a potential route for exposure for humans. Historically, this route has been used extensively for studies of this nature.

- Rationale for animal assignment: random
- Fasting period before blood sampling for (rat) dam thyroid hormones: not examined

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

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

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 and 6-20 (daily)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries:
The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Ovaries and uterine content:

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

Blood sampling:

not examined

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: not examined

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Indices:

Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group No. Gravid Females/Group

Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%) No. Litters/Group

Postimplantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter No. Implantation Sites/Litter x 100

Summation per Group (%) = Sum of Viable Fetuses Affected/Litter (%)
No. Litters/Group

Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected/Litter No. Viable Fetuses/Litter x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

All females in the control, 10, 40, and 160 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. No test substance-related clinical findings were noted at the daily examinations or 1 hour following dose administration at any dosage level. Findings noted in the treated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean maternal body weight gains in the 160 mg/kg/day group were similar to the control group during gestation days 6-9, 9-12, and 12-15. Test substance-related lower mean body weight gains were noted in this group compared to the control group during the gestation day interval 15-20 due to lower mean body weight gains during gestation days 18-20; the differences were significant (p<0.05 or p<0.01). As a result, mean body weight gain in the 160 mg/kg/day group was 14.2% lower than the control group for the overall treatment period (gestation days 6-20) and mean body weights on gestation days 19 and 20 were 4.9% and 5.4% lower, respectively, compared to the control group; the differences were significant (p<0.05 or p<0.01). In addition, mean net body weight and net body weight gain in this group were significantly (p<0.01) lower than the control
group by 6.0% and 26.3%, respectively. Mean gravid uterine weight in the
160 mg/kg/day group was similar to the control group. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 10 and 40 mg/kg/day groups were unaffected by
test substance administration. The only significant (p<0.05) difference from the control group was a lower mean body weight gain in the 40 mg/kg/day group during gestation day 18-19. The magnitude of the decrement in mean body weight gain in this group was not sufficient to result in lower mean body weights during the remainder of the study and there was no corresponding reduction in food consumption during or prior to this interval. Therefore, this transient lower mean body weight gain was not attributed to the test substance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, lower mean maternal food consumption, evaluated as
g/animal/day and g/kg/day, was noted in the 160 mg/kg/day group compared to the control group during gestation days 6-9, 9-12, and 15-20; the differences were generally significant (p<0.05 or p<0.01). As a result, significantly (p<0.01) lower mean food consumption was noted in this group when the overall treatment period (gestation days 6-20) was evaluated. The lower mean food consumption value in the 160 mg/kg/day group late in gestation corresponded to the lower mean body weight gain. Mean food consumption in the 10 and 40 mg/kg/day groups was unaffected by test substance administration. The only significant (p<0.05) difference from the control group was a lower mean food consumption (g/animal/day only) during gestation day 19-20. Because mean body weight on gestation day 20 in this group was similar to the control group, the lower mean food consumption in the 40 mg/kg/day group at the end of the treatment period was not considered test substance-related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 10, 40, and 160 mg/kg/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently and in a manner that was not dose-related. Two and 1 females in the control and 40 mg/kg/day groups, respectively, were nongravid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 10, 40, and 160 mg/kg/day. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not examined

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

40

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Two (2) and 1(1) fetuses (litters) in the 10 and 160 mg/kg/day groups, respectively, had external malformations. Fetus no. 21036-14 in the 160 mg/kg/day group had omphalocele (several loops of intestine protruded through an opening in the umbilicus, with remnants of a membranous sac). In the 10 mg/kg/day group, fetus no. 21069-10 had microphthalmia and anophthalmia; there was no apparent skeletal origin for these findings. In addition, fetus no. 21072-14 in the 10 mg/kg/day group had localized edema (thorax). These malformations were considered not to be test substance-related because they occurred in single fetuses, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and the values were within the WIL Research historical control data ranges. No other external malformations were observed in this study. No external developmental variations were noted at any dosage level.

Skeletal malformations:

no effects observed

Description (incidence and severity):

One fetus in the 10 mg/kg/day group had a skeletal malformation. Fetus no. 21072-14 in the 10 mg/kg/day group had a costal cartilage anomaly (fused and malpositioned costal cartilages). This fetus also had localized edema and hydrocephaly. Because the costal cartilage anomaly occurred only in the low-dosage group, the mean litter proportion was not statistically significant from the concurrent control group, and the value was within the WIL Research historical control data range, this malformation was not attributed to the test substance.
No test substance-related skeletal developmental variations were noted at any dosage level. Findings noted in the 10, 40, and/or 160 mg/kg/day groups were observed in single fetuses; occurred similarly in the control group; were not dose-related; and the mean litter proportions were not statistically significantly different from the concurrent control group and/or were within the range of the WIL Research developmental historical control ranges.

Visceral malformations:

no effects observed

Description (incidence and severity):

One fetus each in the 10 and 40 mg/kg/day groups had visceral malformations. Fetus no. 21115-07 in the 40 mg/kg/day group had situs inversus (lateral transposition of the trachea, esophagus, heart, lungs, great and major vessels, stomach, liver, pancreas, spleen, kidney, adrenal glands, and intestine). Fetus no. 21072-14 in the 10 mg/kg/day group had hydrocephaly (increased cavitation of both lateral and the third ventricles); this fetus also had localized edema externally. These malformations occurred in single fetuses, were not observed at the highest dosage level, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and the values were within the WIL Research historical control data ranges. Therefore, the visceral malformations noted in the 10 and 40 mg/kg/day groups were not attributed to the test substance.
No test substance-related visceral developmental variations were noted at any dosage level. Findings noted in the 10, 40, and/or 160 mg/kg/day groups were observed in single fetuses; occurred similarly in the control group; were not noted in a dose-related manner; and the mean litter proportions were not statistically significantly different from the concurrent control group and/or were within the range of the WIL Research developmental historical control ranges. Renal papilla(e) that were not fully developed (Woo and Hoar grade 1) were noted in 5(3) fetuses (litters) in the control group. In addition, fetus no 21097-05 in the 40 mg/kg/day group had white discoloration in the thymus gland. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were considered not to be test substance-related because they occurred infrequently, only in the control group, and/or in a manner that was not dose-related.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on test substance-related effects on mean body weight gain and food consumption at 160 mg/kg/day, a dosage level of 40 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. No test
substance-related effects on intrauterine growth and survival or fetal morphology were noted at any dosage level. Therefore, a dosage level of 160 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for embryo/fetal development when mancozeb was administered orally by gavage to bred Crl:CD(SD) rats. There was no evidence of teratogenicity due to mancozeb in this study.

Executive summary:

The objectives of the study were to determine the potential of the test substance, mancozeb, to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity.

The test substance, mancozeb, in the vehicle (1.0% [w/v] methylcellulose in deionized water) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 10, 40, and 160 mg/kg/day administered at a dosage volume of 10 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females survived to the scheduled necropsy on gestation day 20. There were no test substance-related clinical or macroscopic findings observed at any dosage level. Lower mean body weight gains were noted in the 160 mg/kg/day group near the end of the treatment period, resulting in a lower overall mean body weight gain (14.2% for gestation days 6-20), lower mean body weights at the end of the treatment period (gestation days 19 and 20), net body weight, and net body weight gain compared to the control group. Mean food consumption in this group was generally lower throughout the treatment period. Mean gravid uterine weight in the 160 mg/kg/day group was similar to the control group. No test substance-related effects on mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, or food consumption were noted in the 10 and 40 mg/kg/day groups. Intrauterine growth and survival were not affected by the test substance at any dosage level. There were no test substance-related fetal malformations or developmental variations in the 10, 40, or 160 mg/kg/day groups. Based on test substance-related effects on mean body weight gain and food consumption at 160 mg/kg/day, a dosage level of 40 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. No test substance-related effects on intrauterine growth and survival or fetal morphology were noted at any dosage level. Therefore, a dosage level of 160 mg/kg/day, the highest dosage level tested, was considered to be the NOAEL for embryo/fetal development when mancozeb was administered orally by gavage to bred Crl:CD(SD) rats. There was no evidence of teratogenicity due to mancozeb in this study.