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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-08-06 to 1988-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-08-06 to 1988-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4200 (Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, New York
- Age at study initiation: 37 days
- Housing: in stainless steel, wire-mesh cages; males and females were housed separately; initially three animals/cage during acclimation during the pretest period, individually during the study
- Diet: irradiated Purina certified Rodent Chow (#5002) ad libitum, diets were prepared by adding the appropriate amount of test substance
- Water: tap water ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY:
On the basis of data specified by the manufacturer of the rodent chow, dietary contaminants were determined to be within acceptable ranges. Tap water at Haskell Laboratory was supplied by the Wilmington Suburban Water Corporation (WSWC). Analyses conducted by the water company have shown the concentration of contaminants to be within acceptable ranges. Haskell Laboratory investigations confirmed this result.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/-2
- Humidity (%): 50+/-10
- Photoperiod (h dark / h light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina certified Rodent Chow (#5002)
- Storage temperature of food: up to one week at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To determine the homogeneity of the compound in the diet, samples of the test diet were collected from the top, middle and bottom levels of the mixing vessel. These samples were taken during apportionment of diet storage containers and were frozen immediately. Homogeneity samples were taken for initial mixes of each set of design concentrations: from 0, 10, 30, 65 and 375 ppm diets on test day -1, from 0, 15, 45, 95 and 565 ppm diets on test day 13 and from 0, 20, 60, 125 and 750 ppm diets on test day 27. Stability samples were collected following diet preparation on test days -1, 27, and 174. Samples were taken from diet storage buckets six days after diets were prepared and from rat feeders in the animals room eight days after diet preparation for each of these sampling periods. Following diet preparation on day 174, two-, four-, and six-day feeder samples were also collected. Once each month, one sample was taken from the middle of the mixing vessel for each diet concentration and frozen immediately. During the first year of the study, all of these monthly concentration samples that were collected were submitted for analyses. During the second year, randomly selected samples from those collected once each month were submitted. A sample of freshly prepared control diet was also collected each time diet samples were taken. Samples were frozen after collection and sent to Enviro-Bio-Tech Ltd., Bernville, Pennsylvania for analyses. Whenever samples were taken, a backup sample from one diet storage container in each diet concentration was collected and frozen on the day after collection of the samples. These backup samples and all monthly samples not selected for submittal were retained at Haskell Laboratory, so they would be available to conform analytical results, if necessary. All diet samples were discarded when analytical results became available.

Duration of treatment / exposure:

two years

Frequency of treatment:

daily via feed

Dose / conc.:

0 ppm

Dose / conc.:

20 ppm

Remarks:

corresponding to 0.769 mg/kg bw/d in males and 1.06 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

Dose / conc.:

60 ppm

Remarks:

corresponding to 2.33 mg/kg bw/d in males and 3.06 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

Dose / conc.:

125 ppm

Remarks:

corresponding to 4.83 mg/kg bw/d in males and 6.72 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

Dose / conc.:

750 ppm

Remarks:

corresponding to 30.9 mg/kg bw/d in males and 40.2 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

No. of animals per sex per dose:

72

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on previous 90-day study

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 6 months and biweekly thereafter

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


FOOD EFFICIENCY:
- Food consumption and Body weight data were used to calculate the mean individual food efficiency.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During pretest period and prior to study completion
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18 and 24-month intervals
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes, approximately 16 hours
- How many animals: 10 animals/sex/dose group
- Parameters examined: number of erythrocytes, leukocytes, platelets, hemoglobin concentration, hematocrit, relative number of leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18 and 24-month intervals
- Animals fasted: Yes, approximately 16 hours
- How many animals: 10 animals/sex/dose group
- Parameters examined: activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and creatine kinase; concentrations of urea nitrogen, total serum protein, albumin, creatinine, cholesterol, glucose, calcium, sodium and potassium. Serum globulin concentrations were calculated from the total protein and albumin concentrations.

PLASMA/SERUM HORMONES/LIPIDS: Yes (thyroid hormones)
- Time of blood sample collection: at 3, 6, 12, 18 and 24-month intervals
- Animals fasted: Yes, approximately 16 hours
- How many animals: 10 animals/sex/dose group


URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 6, 12, 18 and 24-month intervals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Urin samples were collected during the 16 hours fasting period prior to blood collection
- How many animals: 10 animals/sex/dose group
- Parameters examined: volume, pH, urobilinogen, osmolality, glucose, protein, bilirubin, ketone, occult blood, colour, transparency, sediment (microscopically examination)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Body weight gains, organ weights and clinical laboratory measurements were analyzed by a one-way analysis of variance. When the test for differences among the test group means (F test) was significant, pairwise comparisons were made between test and control groups. For body weight gains and clinical laboratory measurements these comparisons were made with the Dunnett's test. Organ weights comparisons were made with the Least Square Differences (LSD) and Dunnett's tests. The incidences of clinical observations were valuated by Fisher's Exact test with a Bonferroni correction and the Cochran-Armitage test for trend was used when pairwise comparisons were significant. Histopathology lesion incidences were valuated with the Fisher's Exact Test. Significance was judged at alpha= 0.05. Independent statistical analyses were conducted for each sex.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males of the high dose group had a statistically significant low incidence of diarrhea. Females of the same dose group had a statistically not significant low incidence of diarrhea.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Unscheduled mortalities for rats in this study could not be directly attributed to the dietary intake of the test material. Survival rates were generally lower for male rats than for female rats by the end of the two-year feeding period (Day 728). The percent survival for rats fed 0, 20, 60, 125, or 750 ppm test item in study diets was 26%, 29%, 39%, 16%, and 21%, respectively, for males and 37%, 53%, 39%, 39%, and 42% for females. Although male survival rates were low in this study, they were considered acceptable, given the range of survival rates of male controls (27-34%) in four other studies recently conducted at this laboratory.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of male rats fed diets that contained 750 ppm test item were lower throughout the study than the mean body weights of male control rats. By day 364, mean body weight of males fed 750 ppm diets was 7.4% lower compared to control rats. During the first three months of the study (days 0-91), female rats of the highest dose group had a mean body weight that was more than 10% lower than the mean body weight of female control rats and a statistically significant lower body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean food efficiency for male and female rats fed 750 ppm diets was lower than the food efficiency for respective control groups during the first 91 days of the study. The mean food efficiency values were similar for all groups within a sex over the entire study. The results indicate that there were compound-related effects on utilization of food for body weight gain during the first three months; however, these differences were not apparent when measured over the two-year period.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Prior to completion of the study, the rats that remained alive were examined on test day 702. At that time, 15 males and 15 females had ocular lesions. Pale ocular fundi was the most frequent finding; however, these lesions are often due to anemia, and not to primary ocular disease. Other observed ocular lesions included cataracts, vascularization, hemorrhage, and phthisis. The eye lesions occurred among all study groups and were considered not to have been changes induced by dietary intake of the test material.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum sodium results in male and female rats in several treatment groups appeared low at the 3-month sampling period; however, the effect did not occur at later sampling times. In addition, the sodium levels for these groups were higher than controls at 24 months. Since the low sodium was not a consistent finding, the 3-month levels were considered not to be related to compound intake. There were a number of other statistically significant findings noted at individual sampling points but these results either were within the range of control values at this laboratory or did not occur consistently and were considered spurious or age-related effects.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

The results of endocrine assays indicated that intake of the test item had an effect on hormones associated with thyroid function in male and female rats fed diets that contained 750 ppm of the test material. Low levels of thyroxine (T4, up to 52 % compared to control) and elevated levels of thyroid stimulating hormone (TSH, up to 113 % compared to control) were evident in the plasma at repeated sampling points throughout the study. If considered alone, the levels of T4 may not have been interpreted as biologically important; however, the known relationship between elevated serum TSH and low T4 levels indicate a compound-related effect exists in the rats fed 750 ppm diets. Triiodothyronine (T3) levels were low and statistically significant for several groups at the 3-month sampling point, but this finding was not consistent throughout the study. It was also noted that T3 levels were not affected in rats fed 1000 ppm test item diets in a previously conducted 90-day study. Therefore, the alterations in T3- levels could not be linked to dietary consumption of the test material. No compound-related hormone effects were noted in rats fed diets that contained 125 ppm or less of the test item.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male and female rats of the highest dose group had elevated mean absolute and relative thyroid/parathyroid weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

When results were summarized for the first year, there were no statistically significant differences in incidences of gross observations. During the second year of the study there were a number of statistically significant differences observed. Many findings were incidental; however, a high incidence of enlarged thyroids observed among both male and female rats fed diets that contained 750 ppm mancozeb and thyroid masses among the males in that dietary concentration group were considered related to dietary intake of mancozeb.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During the histopathological evaluation of tissues from rats that died or were sacrificed during the first year of the study, there were increased incidences of thyroid effects in male and female rats fed 750 ppm mancozeb diets. The changes included slight to moderate diffuse colloid depletion, a decrease in follicle size, and/or follicular cell hypertrophy. High dose male and female rats evaluated during the second year of the study had increased incidences of nodular hyperplasia and hypertrophy/hyperplasia. Changes were also noted during the second year in the eyes of rats fed 750 ppm diets. An increase in incidence of bilateral retinopathy was observed in male and female rats. Although retinopathy is a common lesion among aging rats, the lesion severity was generally greater in this dietary group than that noted for the other study groups. The increased incidence and severity of retinopathy was considered a compound-related effect.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During the second year, male and female rats fed 750 ppm diets had increased incidences of thyroid follicular cell carcinomas and adenomas. There were 14 rats with follicular cell carcinomas and 20 with follicular cell adenomas among 61 male rats examined from this group during the second year of the study. None of the male control rats examined during the same period exhibited either of these tumor types. Six male rats in this highest dietary group had both carcinomas and adenomas. In 61 females examined from the group fed 750 ppm diets, there were four with follicular cell carcinomas and six with adenomas. One female rat from the control group had a follicular cell adenoma and none of the female controls exhibited follicular cell carcinoma. The number of female rats from the 750 ppm dietary concentration group with thyroid carcinomas was not statistically significant, but the incidence was considered biologically important.

Details on results:

Body weight:
The mean body weights of male rats fed diets that contained 750 ppm test item were lower throughout the study than the mean body weights of male control rats. Among male rats that survived the entire study, the mean body weight gains for those groups fed diets that contained the test material were not statistically significant when compared to the group fed control diet. During the first year, however, male rats fed 750 ppm diets had a statistically significant lower mean body weight gain (day 0-364). By day 364, mean body weight of males fed 750 ppm diets was 739.1 grams. This was 7.4% lower than the mean body weight of 798 grams exhibited by male control rats. The difference was considered to be a compound-related effect. None of the other groups of male rats exhibited body weight effects related to dietary intake of test item. From test day 532 to the end of the study, individual weight losses occurred for many study rats, and this resulted in negative gains for all groups. These weight losses were attributed to aging and not to compound intake. During the first three months of the study (days 0-91), female rats fed diets that contained 750 ppm of test compound had a mean body weight that was more than 10% lower than the mean body weight of female control rats. Between days 0 and 91, this group also had a statistically significant lower body weight gain than the female control rats. This low initial weight gain was considered related to dietary intake of test item. After the first three months, however, the weight gain effects are not as clear. The female rats fed 750 ppm diets showed an apparent recovery from initial effects on weight, and females fed 20 or 125 ppm diets had lower mean weight gains than the female controls. By the end of the first 12 months of the study, female rats in the groups fed 20, 125, and 750 ppm diets all had mean body weight gains that were lower than the mean body weight gains of female control rats (days 0-364). These weight gains during the first year were statistically significant; however, there was no dose-response relationship evident, and the low gains could not be attributed to compound intake. Weight gains for all compound-treated female groups were similar to those of controls for rats that survived for two years. Consistent weight losses were evident for all female groups during the last months of the study. With the exception of the initial (0-91 days) low weight gain by females in the 750 ppm group, the body weight effects in females could not be attributed to dietary intake of the test material.
Clinical observations:
There were several statistically significant incidences of clinical signs among male rats in this study. Low incidences of lethargy were noted for rats fed 60 or 125 ppm test item diets; however, the biological significance of this observation is not clear. Rats fed 750 ppm test item diets had a statistically significant low incidence of diarrhea. No significant incidences were noted in total palpable masses for any group of male rats when compared to the male controls. Female rats had very few statistically significant incidences of clinical signs. Female rats in the 750 ppm diet group had a low incidence of wet fur, in the 125 ppm group a low incidence of stained fur, and in the 60 ppm group a high incidence of swollen inguen. None of these observations could be attributed to compound intake. Although not statistically significant, female rats in the group fed 750 ppm diets had a low incidence of diarrhea. Coupled with the low incidence of male rats with diarrhea, it appears this is an effect related to dietary intake of test material. The total numbers of palpable masses were similar for all female groups, although the group fed 60 ppm diets had a statistically significant high incidence of clinically observed abdominal masses. This high number of masses was considered incidental and not related to compound intake.
Clinical Laboratory Evaluations:
None of the hematology, clinical chemistry, or urinalysis results obtained in this study showed evidence of effects associated with the dietary intake of the test substance. Serum sodium results in male and female rats in several treatment groups appeared low at the 3-month sampling period; however, the effect did not occur at later sampling times. In addition, the sodium levels for these groups were higher than controls at 24 months. Since the low sodium was not a consistent finding, the 3-month levels were considered not to be related to compound intake. There were a number of other statistically significant findings noted at individual sampling points but these results either were within the range of control values at this laboratory or did not occur consistently and were considered spurious or age-related effects. The results of endocrine assays indicated that intake of the test item had an effect on hormones associated with thyroid function in male and female rats fed diets that contained 750 ppm of the test material. Low levels of thyroxine (T4) and elevated levels of thyroid stimulating hormone (TSH) were evident in the plasma at repeated sampling points throughout the study. If considered alone, the levels of T4 may not have been interpreted as biologically important; however, the known relationship between elevated serum TSH and low T4 levels indicate a compound-related effect exists in the rats fed 750 ppm diets. Triiodothyronine (T3) levels were low and statistically significant for several groups at the 3-month sampling point, but this finding was not consistent throughout the study. It was also noted that T3 levels were not affected in rats fed 1000 ppm test item diets in a previously conducted 90-day study. Therefore, the alterations in T3- levels could not be linked to dietary consumption of the test material. No compound-related hormone effects were noted in rats fed diets that contained 125 ppm or less of the test item.
Pathological Evaluation:
Selected organs were weighed during the 12-month and 24-month scheduled sacrifices of study rats. At the twelve-month sacrifice, the mean relative liver and testes weights were elevated among male rats fed diets that contained 750 ppm mancozeb. Although the relative weights of these organs were statistically significant, the absolute weights were not, and the significance was considered to be due to low body weights of the male rats in this group, not to toxic effects from dietary intake of mancozeb. A low mean absolute thyroid/parathyroid weight for female rats fed 20 ppm diets was ; considered spurious and not a compound-related effect. At the 24-month sacrifice, male rats fed 750 ppm diets had an elevated mean relative liver weight and male and female rats had elevated mean absolute and relative thyroid/parathyroid weights. The mean absolute liver weight for male rats fed 750 ppm diets was not statistically significant and the high mean relative liver weight was considered a result of the low mean total body weight of the rats in this group. The elevations in thyroid/parathyroid weight for the male and female rats were considered compound-related effects. All rats in the study were examined grossly at necropsy. When results were summarized for the first year, there were no statistically significant differences in incidences of gross observations. During the second year of the study there were a number of statistically significant differences observed. Many findings were incidental; however, a high incidence of enlarged thyroids observed among both male and female rats fed diets that contained 750 ppm mancozeb and thyroid masses among the males in that dietary concentration group were considered related to dietary intake of mancozeb. During the histopathological evaluation of tissues from rats that died or were sacrificed during the first year of the study, there were increased incidences of thyroid effects in male and female rats fed 750 ppm mancozeb diets. The changes included slight to moderate diffuse colloid depletion, a decrease in follicle size, and/or follicular cell hypertrophy. Rats evaluated during the second year of the study exhibited additional histopathological effects in the thyroid glands. Male and female rats fed 750 ppm diets had increased incidences of thyroid follicular cell carcinomas, adenomas, nodular hyperplasia and hypertrophy/hyperplasia. There were 14 rats with follicular cell carcinomas and 20 with follicular cell adenomas among 61 male rats examined from this group during the second year of the study. None of the male control rats examined during the same period exhibited either of these tumor types. Six male rats in this highest dietary group had both carcinomas and adenomas. In 61 females examined from the group fed 750 ppm diets, there were four with follicular cell carcinomas and six with adenomas. One female rat from the control group had a follicular cell adenoma and none of the female controls exhibited follicular cell carcinoma. The number of female rats from the 750 ppm dietary concentration group with thyroid carcinomas was not statistically significant, but the incidence was considered biologically important. None of the other groups fed diets that contained mancozeb had any significant histopathological changes in the thyroid glands. Male and female groups that exhibited thyroid carcinomas and adenomas also had low levels of thyroxine (T4) and elevated levels of thyroid stimulating hormone (TSH). The relationship between presence of follicular cell neoplasia and serum levels of T4 and TSH is indicative of a hormonally-mediated mechanism of carcinogenicity. Prolonged stimulation of the thyroid-pituitary feedback mechanism and resultant release of elevated levels of TSH are thought to lead to thyroid gland neoplasia in rats. In studies conducted in several animal species with a number of different goitrogens, rats appear to be the species which is most sensitive to thyroid tumor formation, and no evidence exists for the occurrence of thyroid carcinogenicity by this mechanism in humans. The histopathological changes seen here are similar to those observed in a two-year rat oncogenicity study conducted with ethylene thiourea (ETU). Since ETU is a metabolite of mancozeb, these follicular cell adenomas and carcinomas are considered related to the presence of ETU. ETU belongs to a class of compounds which inhibit thyroid hormone synthesis by inhibition of iodine peroxidase. At least one study in rats has shown some reversibility of early thyroid effects induced by ETU. The indicators for a hormonally-induced mechanism of carcinogenesis and the absence of follicular cell neoplasia in rats fed dietary concentrations of 125 ppm mancozeb or lower suggests a threshold for induction of follicular cell neoplasia in this study. Changes were also noted during the second year in the eyes of rats fed 750 ppm diets. An increase in incidence of bilateral retinopathy was observed in male and female rats. Although retinopathy is a common lesion among aging rats, the lesion severity was generally greater in this dietary group than that noted for the other study groups. The increased incidence and severity of retinopathy was considered a compound-related effect. There was also a dose-dependent incidence of rats with a glassy, yellow-brown pigment in the cortical tubular lumina of the kidneys in the 750 and 125 ppm diet groups of both sexes. Although the presence of the pigment was considered related to the intake of test compound, no toxic effects could be associated with its presence. No pathological changes were associated with the pigment and none of the clinical chemistry parameters generally associated with kidney function indicated adverse renal effects in these groups. None of the other statistically significant incidences of histopathological lesions could be attributed to the dietary intake of test material. Absence of findings in the liver correlated with the conclusion that liver weight differences were incidental and not compound related. Lesions observed were considered either incidental occurrences or age- or disease-related lesions, and the incidences were within normal biological variation for this strain of rats.

Key result

Dose descriptor:

NOAEL

Effect level:

4.8 other: mg/kg bw/d

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of effects

Key result

Dose descriptor:

NOAEL

Effect level:

6.7 other: mg/kg bw/d

Based on:

test mat.

Sex:

female

Basis for effect level:

other: absence of effects

Dose descriptor:

LOAEL

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: 750 ppm in diet corresponds to a test substance concentration of 30.9 mg/kg bw/d for males and 40.2 mg/kg bw/d for females

The tables below are taken from the Draft (Renewal) Assessment Report prepared according to the Commission Regulation (EU) N° 1107/2009 (Volume 3-B.6) of Mancozeb:

 

Table 1: Intergroup comparison of thyroxine (T4, μg/dL), thyroid stimulating hormone (TSH ng/mL) and triiodothyronine (T3 ng/dL) at selected timepoints

Parameter		Dietary concentration (ppm)
		Males					Females
	Month	0	20	60	125	750	0	20	60	125	750
T3	3	110	128	114	82*	77*	116	105	91*	82*	80*
T3	6	90	104	89	110	111*	114	126	114	124	120
T3	24	47	66	65	57	67#	69	68	78	79	86
T4	3	3.6	4.2	4.5*	4.3	3.0	2.2	2.3	2.0	2.2	1.1*
T4	6	3.8	4.1	4.1	3.8	3.1#	2.3	2.5	2.8	2.6	1.9
T4	18	3.1	2.8	3.4	3.1	2.0*	2.7	2.1*	2.5	2.7	2.1*
T4	24	1.8	2.6	1.5	1.4	1.8	1.4	1.7	1.5	1.9	1.1#
TSH	6	4.8	5.1	3.1	3.8	6.2	3.4	3.6	3.3	3.3	6.0*
TSH	12	3.0	2.7	1.9*	2.2	4.0*	3.0	2.8	2.6	2.6	3.5
TSH	18	3.6	2.5	2.5	2.8	5.5*	2.2	2.2	3.1	2.9	4.7#

* Statistically significant difference from control group mean, p<0.05 Dunnett test

# Statistically significant difference from control group mean, p<0.05 Mann Whitney U test

 

Table 2: Intergroup comparison of thyroid weights (g) and relative thyroid weights at 24 months

	Dietary concentration (ppm)
	Males					Females
	0	20	60	125	750	0	20	60	125	750
Absolute	0.047	0.045	0.049	0.052	0.075*	0.037	0.040	0.036	0.041	0.053*
Relative	0.0073	0.0064	0.0070	0.0071	0.0118*	0.0078	0.0075	0.0078	0.0085	0.0115*

* Statistically significant difference from control group mean, p<0.05 Dunnett test

 

Table 3: Intergroup comparison of microscopic non-neoplastic findings day 366 to 737

		Males					Females
Dietary concentrations (ppm)		0	20	60	125	750	0	20	60	125	750
Number examined		60	62	61	58	61	62	60	62	61	61
Thyroid follicular cell	Hyperplasia nodular	0	1	3	2	15*	1	2	2	0	11*
	Hyperplasia/hypertrophy	1	1	2	1	34*	1	0	1	0	24*
Eye	Bilateral retinopathy	4	2	1	3	19*	21	28	24	31*	49*

* Statistically significant difference from control group mean, p<0.05 Fischer’s

 

Table 4: Intergroup comparison of microscopic neoplastic findings day 366 to 737

		Males					Females
Dietary concentration (ppm)		0	20	60	125	750	0	20	60	125	750
Number examined		60	62	61	58	61	62	60	62	61	61
Thyroid follicular cell	Adenoma	0	1	1	0	20*	1	1	1	1	6
	Carcinoma	0	0	2	2	14*	0	0	0	1	4

* Statistically significant difference from control group mean, p<0.05 Fischer’s

 

Conclusions:

Based on the results of this two-year combined chronic toxicity study/carcinogenicity study, the NOAEL of the test substance was determined to be 4.8 mg/kg bw/d.

Executive summary:

In this combined chronic toxicity study/carcinogenicity study, five groups of 72 male and five groups of 72 female Crl:CD®BR rats were fed diets that contained 0, 20, 60, 125, or 750 ppm mancozeb. The rats were weighed at regular intervals and food consumption was monitored throughout the study. Hematology, clinical chemistry, urine, and thyroid hormone assays were conducted approximately 3, 6, 12, 18 and 24 months after study initiation. At 12 months, 10 rats from each group were sacrificed and at 24 months, all surviving rats were sacrificed. All rats that died or were sacrificed during the study were necropsied, and selected tissues were evaluated microscopically.

 

The male rats fed diets that contained 750 ppm mancozeb had a statistically significant low mean body weight gain during the first year of the study when they were compared to the male control rats. Although the statistically significant difference in weight gain did not persist, a lower mean body weight was evident for this group throughout the second year of the study. This difference was attributed to intake of the test material. The female rats fed 750 ppm diets had an initial statistically significant low body weight gain (days 0-91) that was considered a compound-related effect. After the first three months, this group showed an apparent recovery, illustrated by body weight gains that were similar to those in other treated groups. Over the duration of the first year, body weight gains for the 20, 125, and 750 ppm groups of females were lower than female controls, but these differences could not be directly attributed to the dietary intake of mancozeb.

 

There were no compound-related differences in mean daily food consumption or mean food efficiency. In the male groups fed diets that contained 0, 20, 60, 125, or 750 ppm mancozeb, mean daily intake during the study was 0, 0.769, 2.33, 4.83, and 30.9 mg mancozeb/kg body weight/day, respectively. Mean daily intake by female rats in the same respective dietary concentration groups was 0, 1.06, 3.06, 6.72, and 40.2 mg mancozeb/kg body weight/day.

 

A low incidence of diarrhea among male rats fed 750 ppm mancozeb diets was the only clinical sign attributed to compound intake in rats in this study. None of the lesions observed during the ophthalmological examination of rats at the end of the study were considered compound related.

 

Early deaths among rats in the study were not attributed to compound-related effects. In general, male survival rates were lower than those for female rats. The percent survival for male rats fed 0, 20, 60, 125, or 750 ppm mancozeb in study diets was 26%, 29%, 39%, 16%, and 21%, respectively, and for female rats fed the same dietary levels was 37%, 53%, 39%, 39%, and 42%.

 

Clinical laboratory evaluations that included hematology, clinical chemistry, and urinalyses showed no compound-related effects in rats during the study. Two parameters that measure thyroid function were statistically different from controls in both male and female rats fed 750 ppm diets. Mean thyroxine (T4) levels were lower than T4 levels of controls and levels of thyroid stimulating hormone (TSH) were higher in these groups than in respective male and female control groups.

 

At the 12-month sacrifice, statistically significant elevated mean relative liver and testes weights were seen in male rats fed 750 ppm diets. The mean absolute weights of these organs were similar to the mean liver and testes weights from the male controls; therefore, the increased relative weights were attributed to the lower whole body weights of male rats in this dietary concentration group. None of the gross pathology observations during the 12-month necropsy were statistically significant; however, several significant microscopic observations were noted. Follicular cell hypertrophy/hyperplasia of the thyroid gland was visible in male and female rats fed diets that contained 750 ppm mancozeb. Changes appeared more severe in males than females. No thyroid changes were observed in rats fed dietary concentrations of 125 ppm or lower. Granular pigment was visible microscopically in the kidneys of male and female rats fed 125 ppm or 750 ppm diets, but no tissue alteration was associated with the occurrence of this pigment. At the 24-month sacrifice and necropsy, male rats fed 750 ppm diets had an elevated mean relative liver weight, and male and female rats fed these diets had elevated mean absolute and relative thyroid/parathyroid weights. The high relative liver weight was considered due to the low mean body weight for this group. The elevated thyroid weights were attributed to intake of mancozeb.

 

Gross changes observed during the necropsy of rats during the second year of the study included enlarged thyroids in both males and females fed 750 ppm diets and thyroid masses in the male rats in this dietary group.

 

A number of compound-related histopathological changes were observed in tissues from rats that died or were sacrificed during the second year of the study. Male and female rats fed 750 ppm diets had increased incidences of thyroid follicular cell carcinomas, adenomas, nodular hyperplasia and hypertrophy/hyperplasia. The incidences of rats with carcinomas or adenomas were higher for males (14/61 and 20/61, respectively) than for females (4/61 and 6/61). These thyroid changes are considered to be related to ethylene thiourea (ETU), a known thyroid carcinogen and product of mancozeb metabolism in the rat. ETU belongs to a class of compounds that inhibits synthesis of thyroid hormone and induces release of high TSH levels by the pituitary. Thyroid follicular cell adenomas and carcinomas are believed to result when the threshold for the thyroid-pituitary feedback mechanism is exceeded.

 

Changes were also evident during microscopic evaluation of the eyes of rats fed 750 ppm diets. Increased incidences and severity of bilateral retinopathy were noted for both male and female rats. The yellow-brown pigment in the kidney, noted during examination of the rats from the 12-month sacrifice, was also noted in rats in the 125 and 750 ppm dietary groups during the second year of the study. No tissue pathology was associated with the pigment. Although the presence of this pigment appeared to result from the intake of test material, it was determined not to have been related to any adverse biological effect.

 

The notable toxic effects attributed to the dietary intake of test material for the study were seen in rats fed diets that contained 750 ppm mancozeb. These effects included low body weight gains, alterations in thyroid hormone levels, increased thyroid weights, thyroid follicular cell hypertrophy/hyperplasia, thyroid adenomas, thyroid carcinomas, and bilateral retinopathy. The no-observable-adverse-effect level for rats fed diets that contained mancozeb during the study was 125 ppm (which corresponds to 4.8 mg mancozeb/kg bw/d in males and 6.7 mg mancozeb/kg bw/d in females.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA 870.4200

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Remarks:

US. EPA GLP regulations title 40 CFR part 160 equivalent to OECD GLP Principles, 1982.

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 animals

Species:

rat

Strain:

other: Crl: CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 37 days
- Housing: in stainless steel, wire-mesh cages; males and females were housed seperately; initially three animals/cage during acclimation during the pretest period, individually during the study
- Diet: irridiated Purina certified Rodent Chow (#5002) ad libitum, diets were prepared by adding the appropriate amount of test substance
- Water: tap water ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY:
On the basis of data specified by the manufacturer of the rodent chow, dietary contaminants were determined to be within acceptable ranges. Tap water at Haskell Laboratory was supplied by the Wilmington Suburban Water Corporation (WSWC). Analyses conducted by the water company have shown the concentration of contaminants to be within acceptable ranges. Haskell Laboratory investigations confirmed this result.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/-2
- Humidity (%): 50+/-10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Due to the potential for degradation of the test material upon exposure to moisture or light, the compound was received in individual, tightly-capped containers that held a quantity sufficient for diet preparation each week. When these containers were opened, the compound was used for diet praparation and excess test material was discarded.
The test material was added to the feed and thoroughly mixed for three minutes. Control diets were mixed in the same way. All diets were prepared weekly and stored at room temperature until use.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina certified Rodent Chow (#5002)
- Storage temperature of food: up to one week at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To determine the homogeneity of the compound in the diet, samples of the test diet were collected from the top, middle and bottom levels of the mixing vessel. These samples were taken during apportionment of diet storage containers and were frozen immediately. Homogeneity samples were taken for initial mixes of each set of design concentrations: from 0, 10, 30, 65 and 375 ppm diets on test day -1, from 0, 15, 45, 95 and 565 ppm diets on test day 13 and from 0, 20, 60, 125 and 750 ppm diets on test day 27.
Stability samples were collected following diet preparation on test days -1, 27, and 174. Samples were taken from diet storage buckets six days after diets were prepared and from rat feeders in the animals room eight days after diet preparation for each of these sampling periods. Following diet preparation on day 174, two-, four-, and six-day feeder samples were also collected. Once each month, one sample was taken from the middle of the mixing vessel for each diet concentration and frozen immediately. During the first year of the study, all of these monthly concentration samples that were collected were submitted for analyses. During the second year, randomly selected samples from those collected once each month were submitted. A sample of freshly prapared control diet was also collected each time diet samples were taken. Samples were frozen after collection and sent to Enviro-Bio-Tech Ltd., Bernville, Pennsylvania for analyses.
Whenever samples were taken, a backup sample from one diet storage container in each diet concentration was collected and frozen on the day after collection of the samples. These backup samples and all monthly samples not selected for submittal were retained at Haskell Laboratory, so they would be available to conform analytical results, if necessary. All diet samples were discarded when analytical results became available.

Duration of treatment / exposure:

two years

Frequency of treatment:

daily via feed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

20 ppm

Remarks:

corresponding to 0.769 mg/kg bw/d in males and 1.06 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

Doses / concentrations 3

Dose / conc.:

60 ppm

Remarks:

corresponding to 2.33 mg/kg bw/d in males and 3.06 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

Doses / concentrations 4

Dose / conc.:

125 ppm

Remarks:

corresponding to 4.83 mg/kg bw/d in males and 6.72 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

Doses / concentrations 5

Dose / conc.:

750 ppm

Remarks:

corresponding to 30.9 mg/kg bw/d in males and 40.2 mg/kg bw/d in females (mean calculated dose rate based on nominal dietary level)

No. of animals per sex per dose:

72

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on previous 90-day study

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 6 months and biweekly thereafter

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

FOOD EFFICIENCY:
- Food consumption and Body weight data were used to calculate the mean individual food efficiency.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During pretest period and prior to study completion
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18 and 24-month intervals
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes, approximately 16 hours
- How many animals: 10 animals/sex/dose group
- Parameters examined: number of erythrocytes, leukocytes, platelets, hemoglobin concentration, hematocrit, relative number of leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18 and 24-month intervals
- Animals fasted: Yes, approximately 16 hours
- How many animals: 10 animals/sex/dose group
- Parameters examined: activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and creatine kinase; concentrations of urea nitrogen, total serum protein, albumin, creatinine, cholesterol, glucose, calcium, sodium and potassium. Serum globulin concentrations were calculated from the total protein and albumin concentrations.

PLASMA/SERUM HORMONES/LIPIDS: Yes (thyroid hormones)
- Time of blood sample collection: at 3, 6, 12, 18 and 24-month intervals
- Animals fasted: Yes, approximately 16 hours
- How many animals: 10 animals/sex/dose group

URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 6, 12, 18 and 24-month intervals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Urin samples were collected during the 16 hours fasting period prior to blood collection
- How many animals: 10 animals/sex/dose group
- Parameters examined: volume, pH, urobilinogen, osmolality, glucose, protein, bilirubin, ketone, occult blood, colour, transparency, sediment (microscopically examination)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Body weight gains, organ weights and clinical laboratory measrurements were analyzed by a one-way analysis of variance. When the test for differences among the test group means (F test) was significant, pairwise comparisons were made between test and control groups. For body weight gains and clinical laboratory measurements these comparisons were made with the Dunnett's test. Organ weights comparisons were made with the Least Square Differences (LSD) and Dunnett's tests.The incidences of clinical observations were valuated by Fisher's Exact test with a Bonferroni correction and the Cochran-Armitage test for trend wa sused when pairwise comparisons were significant. Histopathology lesion incidences were valuated with the Fisher's Exact Test. Significance was judged at alpha= 0.05. Independent statistical analyses were conducted for each sex.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males of the high dose group had a statistically significant low incidence of diarrhea. Females of the same dose group had a statistically not significant low incidence of diarrhea.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Unscheduled mortalities for rats in this study could not be directly attributed to the dietary intake of the test material. Survival rates were generally lower for male rats than for female rats by the end of the two-year feeding period (Day 728). The percent survival for rats fed 0, 20, 60, 125, or 750 ppm test item in study diets was 26%, 29%, 39%, 16%, and 21%, respectively, for males and 37%, 53%, 39%, 39%, and 42% for females. Although male survival rates were low in this study, they were considered acceptable, given the range of survival rates of male controls (27-34%) in four other studies recently conducted at this laboratory.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of male rats fed diets that contained 750 ppm test item were lower throughout the study than the mean body weights of male control rats. By day 364, mean body weight of males fed 750 ppm diets was 7.4% lower compared to control rats. During the first three months of the study (days 0-91), female rats of the highest dose group had a mean body weight that was more than 10% lower than the mean body weight of female control rats and a statistically significant lower body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean food efficiency for male and female rats fed 750 ppm diets was lower than the food efficiency for respective control groups during the first 91 days of the study. The mean food efficiency values were similar for all groups within a sex over the entire study. The results indicate that there were compound-related effects on utilization of food for body weight gain during the first three months; however, these differences were not apparent when measured over the two-year period.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Prior to completion of the study, the rats that remained alive were examined on test day 702. At that time, 15 males and 15 females had ocular lesions. Pale ocular fundi was the most frequent finding; however, these lesions are often due to anemia, and not to primary ocular disease. Other observed ocular lesions included cataracts, vascularization, hemorrhage, and phthisis. The eye lesions occurred among all study groups and were considered not to have been changes induced by dietary intake of the test material.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum sodium results in male and female rats in several treatment groups appeared low at the 3-month sampling period; however, the effect did not occur at later sampling times. In addition, the sodium levels for these groups were higher than controls at 24 months. Since the low sodium was not a consistent finding, the 3-month levels were considered not to be related to compound intake. There were a number of other statistically significant findings noted at individual sampling points but these results either were within the range of control values at this laboratory or did not occur consistently and were considered spurious or age-related effects.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

The results of endocrine assays indicated that intake of the test item had an effect on hormones associated with thyroid function in male and female rats fed diets that contained 750 ppm of the test material. Low levels of thyroxine (T4, up to 52 % compared to control) and elevated levels of thyroid stimulating hormone (TSH, up to 113 % compared to control) were evident in the plasma at repeated sampling points throughout the study. If considered alone, the levels of T4 may not have been interpreted as biologically important; however, the known relationship between elevated serum TSH and low T4 levels indicate a compound-related effect exists in the rats fed 750 ppm diets. Triiodothyronine (T3) levels were low and statistically significant for several groups at the 3-month sampling point, but this finding was not consistent throughout the study. It was also noted that T3 levels were not affected in rats fed 1000 ppm test item diets in a previously conducted 90-day study. Therefore, the alterations in T3- levels could not be linked to dietary consumption of the test material. No compound-related hormone effects were noted in rats fed diets that contained 125 ppm or less of the test item.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male and female rats of the highest dose group had elevated mean absolute and relative thyroid/parathyroid weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

When results were summarized for the first year, there were no statistically significant differences in incidences of gross observations. During the second year of the study there were a number of statistically significant differences observed. Many findings were incidental; however, a high incidence of enlarged thyroids observed among both male and female rats fed diets that contained 750 ppm mancozeb and thyroid masses among the males in that dietary concentration group were considered related to dietary intake of mancozeb.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During the histopathological evaluation of tissues from rats that died or were sacrificed during the first year of the study, there were increased incidences of thyroid effects in male and female rats fed 750 ppm mancozeb diets. The changes included slight to moderate diffuse colloid depletion, a decrease in follicle size, and/or follicular cell hypertrophy. High dose male and female rats evaluated during the second year of the study had increased incidences of nodular hyperplasia and hypertrophy/hyperplasia. Changes were also noted during the second year in the eyes of rats fed 750 ppm diets. An increase in incidence of bilateral retinopathy was observed in male and female rats. Although retinopathy is a common lesion among aging rats, the lesion severity was generally greater in this dietary group than that noted for the other study groups. The increased incidence and severity of retinopathy was considered a compound-related effect.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During the second year, male and female rats fed 750 ppm diets had increased incidences of thyroid follicular cell carcinomas and adenomas. There were 14 rats with follicular cell carcinomas and 20 with follicular cell adenomas among 61 male rats examined from this group during the second year of the study. None of the male control rats examined during the same period exhibited either of these tumor types. Six male rats in this highest dietary group had both carcinomas and adenomas. In 61 females examined from the group fed 750 ppm diets, there were four with follicular cell carcinomas and six with adenomas. One female rat from the control group had a follicular cell adenoma and none of the female controls exhibited follicular cell carcinoma. The number of female rats from the 750 ppm dietary concentration group with thyroid carcinomas was not statistically significant, but the incidence was considered biologically important.

Details on results:

Body weight
The mean body weights of male rats fed diets that contained 750 ppm test item were lower throughout the study than the mean body weights of male control rats. Among male rats that survived the entire study, the mean body weight gains for those groups fed diets that contained the test material were not statistically significant when compared to the group fed control diet. During the first year, however, male rats fed 750 ppm diets had a statistically significant lower mean body weight gain (day 0-364). By day 364, mean body weight of males fed 750 ppm diets was 739.1 grams. This was 7.4% lower than the mean body weight of 798 grams exhibited by male control rats. The difference was considered to be a compound-related effect. None of the other groups of male rats exhibited body weight effects related to dietary intake of test item. From test day 532 to the end of the study, individual weight losses occurred for many study rats, and this resulted in negative gains for all groups. These weight losses were attributed to aging and not to compound intake.
During the first three months of the study (days 0-91), female rats fed diets that contained 750 ppm of test compound had a mean body weight that was more than 10% lower than the mean body weight of female control rats. Between days 0 and 91, this group also had a statistically significant lower body weight gain than the female control rats. This low initial weight gain was considered related to dietary intake of test item. After the first three months, however, the weight gain effects are not as clear. The female rats fed 750 ppm diets showed an apparent recovery from initial effects on weight, and females fed 20 or 125 ppm diets had lower mean weight gains than the female controls. By the end of the first 12 months of the study, female rats in the groups fed 20, 125, and 750 ppm diets all had mean body weight gains that were lower than the mean body weight gains of female control rats (days 0-364). These weight gains during the first year were statistically significant; however, there was no dose-response relationship evident, and the low gains could not be attributed to compound intake. Weight gains for all compound-treated female groups were similar to those of controls for rats that survived for two years. Consistent weight losses were evident for all female groups during the last months of the study. With the exception of the initial (0-91 days) low weight gain by females in the 750 ppm group, the body weight effects in females could not be attributed to dietary intake of the test material.


Clinical observations
There were several statistically significant incidences of clinical signs among male rats in this study. Low incidences of lethargy were noted for rats fed 60 or 125 ppm test item diets; however, the biological significance of this observation is not clear. Rats fed 750 ppm test item diets had a statistically significant low incidence of diarrhea. No significant incidences were noted in total palpable masses for any group of male rats when compared to the male controls.
Female rats had very few statistically significant incidences of clinical signs. Female rats in the 750 ppm diet group had a low incidence of wet fur, in the 125 ppm group a low incidence of stained fur, and in the 60 ppm group a high incidence of swollen inguen. None of these observations could be attributed to compound intake. Although not statistically significant, female rats in the group fed 750 ppm diets had a low incidence of diarrhea. Coupled with the low incidence of male rats with diarrhea, it appears this is an effect related to dietary intake of test material. The total numbers of palpable masses were similar for all female groups, although the group fed 60 ppm diets had a statistically significant high incidence of clinically observed abdominal masses. This high number of masses was considered incidental and not related to compound intake.


Clinical Laboratory Evaluations
None of the hematology, clinical chemistry, or urinalysis results obtained in this study showed evidence of effects associated with the dietary intake of the test substance. Serum sodium results in male and female rats in several treatment groups appeared low at the 3-month sampling period; however, the effect did not occur at later sampling times. In addition, the sodium levels for these groups were higher than controls at 24 months. Since the low sodium was not a consistent finding, the 3-month levels were considered not to be related to compound intake. There were a number of other statistically significant findings noted at individual sampling points but these results either were within the range of control values at this laboratory or did not occur consistently and were considered spurious or age-related effects. The results of endocrine assays indicated that intake of the test item had an effect on hormones associated with thyroid function in male and female rats fed diets that contained 750 ppm of the test material. Low levels of thyroxine (T4) and elevated levels of thyroid stimulating hormone (TSH) were evident in the plasma at repeated sampling points throughout the study. If considered alone, the levels of T4 may not have been interpreted as biologically important; however, the known relationship between elevated serum TSH and low T4 levels indicate a compound-related effect exists in the rats fed 750 ppm diets. Triiodothyronine (T3) levels were low and statistically significant for several groups at the 3-month sampling point, but this finding was not consistent throughout the study. It was also noted that T3 levels were not affected in rats fed 1000 ppm test item diets in a previously conducted 90-day study. Therefore, the alterations in T3- levels could not be linked to dietary consumption of the test material. No compound-related hormone effects were noted in rats fed diets that contained 125 ppm or less of the test item.


Pathological Evaluation
Selected organs were weighed during the 12-month and 24-month scheduled sacrifices of study rats. At the twelve-month sacrifice, the mean relative liver and testes weights were elevated among male rats fed diets that contained 750 ppm mancozeb. Although the relative weights of these organs were statistically significant, the absolute weights were not, and the significance was considered to be due to low body weights of the male rats in this group, not to toxic effects from dietary intake of mancozeb. A low mean absolute thyroid/parathyroid weight for female rats fed 20 ppm diets was ; considered spurious and not a compound-related effect.
At the 24-month sacrifice, male rats fed 750 ppm diets had an elevated mean relative liver weight and male and female rats had elevated mean absolute and relative thyroid/parathyroid weights. The mean absolute liver weight for male rats fed 750 ppm diets was not statistically significant and the high mean relative liver weight was considered a result of the low mean total body weight of the rats in this group. The elevations in thyroid/parathyroid weight for the male and female rats were considered compound-related effects.
All rats in the study were examined grossly at necropsy. When results were summarized for the first year, there were no statistically significant differences in incidences of gross observations. During the second year of the study there were a number of statistically significant differences observed. Many findings were incidental; however, a high incidence of enlarged thyroids observed among both male and female rats fed diets that contained 750 ppm mancozeb and thyroid masses among the males in that dietary concentration group were considered related to dietary intake of mancozeb.
During the histopathological evaluation of tissues from rats that died or were sacrificed during the first year of the study, there were increased incidences of thyroid effects in male and female rats fed 750 ppm mancozeb diets. The changes included slight to moderate diffuse colloid depletion, a decrease in follicle size, and/or follicular cell hypertrophy.
Rats evaluated during the second year of the study exhibited additional histopathological effects in the thyroid glands. Male and female rats fed 750 ppm diets had increased incidences of thyroid follicular cell carcinomas, adenomas, nodular hyperplasia and hypertrophy/hyperplasia. There were 14 rats with follicular cell carcinomas and 20 with follicular cell adenomas among 61 male rats examined from this group during the second year of the study. None of the male control rats examined during the same period exhibited either of these tumor types. Six male rats in this highest dietary group had both carcinomas and adenomas. In 61 females examined from the group fed 750 ppm diets, there were four with follicular cell carcinomas and six with adenomas. One female rat from the control group had a follicular cell adenoma and none of the female controls exhibited follicular cell carcinoma. The number of female rats from the 750 ppm dietary concentration group with thyroid carcinomas was not statistically significant, but the incidence was considered biologically important. None of the other groups fed diets that contained mancozeb had any significant histopathological changes in the thyroid glands. Male and female groups that exhibited thyroid carcinomas and adenomas also had low levels of thyroxine (T4) and elevated levels of thyroid stimulating hormone (TSH).
The relationship between presence of follicular cell neoplasia and serum levels of T4 and TSH is indicative of a hormonally-mediated mechanism of carcinogenicity. Prolonged stimulation of the thyroid-pituitary feedback mechanism and resultant release of elevated levels of TSH are thought to lead to thyroid gland neoplasia in rats. In studies conducted in several animal species with a number of different goitrogens, rats appear to be the species which is most sensitive to thyroid tumor formation, and no evidence exists for the occurrence of thyroid carcinogenicity by this mechanism in humans. The histopathological changes seen here are similar to those observed in a two-year rat oncogenicity study conducted with ethylene thiourea (ETU). Since ETU is a metabolite of mancozeb, these follicular cell adenomas and carcinomas are considered related to the presence of ETU. ETU belongs to a class of compounds which inhibit thyroid hormone synthesis by inhibition of iodine peroxidase. At least one study in rats has shown some reversibility of early thyroid effects induced by ETU.
The indicators for a hormonally-induced mechanism of carcinogenesis and the absence of follicular cell neoplasia in rats fed dietary concentrations of 125 ppm mancozeb or lower suggests a threshold for induction of follicular cell neoplasia in this study.
Changes were also noted during the second year in the eyes of rats fed 750 ppm diets. An increase in incidence of bilateral retinopathy was observed in male and female rats. Although retinopathy is a common lesion among aging rats, the lesion severity was generally greater in this dietary group than that noted for the other study groups. The increased incidence and severity of retinopathy was considered a compound-related effect.
There was also a dose-dependent incidence of rats with a glassy, yellow-brown pigment in the cortical tubular lumina of the kidneys in the 750 and 125 ppm diet groups of both sexes. Although the presence of the pigment was considered related to the intake of test compound, no toxic effects could be associated with its presence. No pathological changes were associated with the pigment and none of the clinical chemistry parameters generally associated with kidney function indicated adverse renal effects in these groups.
None of the other statistically significant incidences of histopathological lesions could be attributed to the dietary intake of test material. Absence of findings in the liver correlated with the conclusion that liver weight differences were incidental and not compound related. Lesions observed were
considered either incidental occurrences or age- or disease-related lesions, and the incidences were within normal biological variation for this strain of rats.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: 750 ppm in diet corresponds to a test substance concentration of 30.9 mg/kg bw/d for males and 40.2 mg/kg bw/d for females

2

Key result

Dose descriptor:

NOAEL

Effect level:

6.7 other: mg/kg bw/d

Based on:

test mat.

Sex:

female

Basis for effect level:

other: absence of effects

3

Key result

Dose descriptor:

NOAEL

Effect level:

4.8 other: mg/kg bw/d

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of effects

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

750 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The tables below are taken from the Draft (Renewal) Assessment Report prepared according to the Commission Regulation (EU) N° 1107/2009 (Volume 3-B.6) of Mancozeb:

 

Table 1: Intergroup comparison of thyroxine (T4, μg/dL), thyroid stimulating hormone (TSH ng/mL) and triiodothyronine (T3 ng/dL) at selected timepoints

Parameter		Dietary concentration (ppm)
		Males					Females
	Month	0	20	60	125	750	0	20	60	125	750
T3	3	110	128	114	82*	77*	116	105	91*	82*	80*
T3	6	90	104	89	110	111*	114	126	114	124	120
T3	24	47	66	65	57	67#	69	68	78	79	86
T4	3	3.6	4.2	4.5*	4.3	3.0	2.2	2.3	2.0	2.2	1.1*
T4	6	3.8	4.1	4.1	3.8	3.1#	2.3	2.5	2.8	2.6	1.9
T4	18	3.1	2.8	3.4	3.1	2.0*	2.7	2.1*	2.5	2.7	2.1*
T4	24	1.8	2.6	1.5	1.4	1.8	1.4	1.7	1.5	1.9	1.1#
TSH	6	4.8	5.1	3.1	3.8	6.2	3.4	3.6	3.3	3.3	6.0*
TSH	12	3.0	2.7	1.9*	2.2	4.0*	3.0	2.8	2.6	2.6	3.5
TSH	18	3.6	2.5	2.5	2.8	5.5*	2.2	2.2	3.1	2.9	4.7#

* Statistically significant difference from control group mean, p<0.05 Dunnett test

# Statistically significant difference from control group mean, p<0.05 Mann Whitney U test

 

Table 2: Intergroup comparison of thyroid weights (g) and relative thyroid weights at 24 months

	Dietary concentration (ppm)
	Males					Females
	0	20	60	125	750	0	20	60	125	750
Absolute	0.047	0.045	0.049	0.052	0.075*	0.037	0.040	0.036	0.041	0.053*
Relative	0.0073	0.0064	0.0070	0.0071	0.0118*	0.0078	0.0075	0.0078	0.0085	0.0115*

* Statistically significant difference from control group mean, p<0.05 Dunnett test

 

Table 3: Intergroup comparison of microscopic non-neoplastic findings day 366 to 737

		Males					Females
Dietary concentrations (ppm)		0	20	60	125	750	0	20	60	125	750
Number examined		60	62	61	58	61	62	60	62	61	61
Thyroid follicular cell	Hyperplasia nodular	0	1	3	2	15*	1	2	2	0	11*
	Hyperplasia/hypertrophy	1	1	2	1	34*	1	0	1	0	24*
Eye	Bilateral retinopathy	4	2	1	3	19*	21	28	24	31*	49*

* Statistically significant difference from control group mean, p<0.05 Fischer’s

 

Table 4: Intergroup comparison of microscopic neoplastic findings day 366 to 737

		Males					Females
Dietary concentration (ppm)		0	20	60	125	750	0	20	60	125	750
Number examined		60	62	61	58	61	62	60	62	61	61
Thyroid follicular cell	Adenoma	0	1	1	0	20*	1	1	1	1	6
	Carcinoma	0	0	2	2	14*	0	0	0	1	4

* Statistically significant difference from control group mean, p<0.05 Fischer’s

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this two-year combined chronic toxicity study/carcinogenicity study, the NOAEL of the test substance was determined to be 4.8 mg/kg bw/d.

Executive summary:

In this combined chronic toxicity study/carcinogenicity study, five groups of 72 male and five groups of 72 female Crl:CD®BR rats were fed diets that contained 0, 20, 60, 125, or 750 ppm mancozeb. The rats were weighed at regular intervals and food consumption was monitored throughout the study. Hematology, clinical chemistry, urine, and thyroid hormone assays were conducted approximately 3, 6, 12, 18 and 24 months after study initiation. At 12 months, 10 rats from each group were sacrificed and at 24 months, all surviving rats were sacrificed. All rats that died or were sacrificed during the study were necropsied, and selected tissues were evaluated microscopically.

The male rats fed diets that contained 750 ppm mancozeb had a statistically significant low mean body weight gain during the first year of the study when they were compared to the male control rats. Although the statistically significant difference in weight gain did not persist, a lower mean body weight was evident for this group throughout the second year of the study. This difference was attributed to intake of the test material. The female rats fed 750 ppm diets had an initial statistically significant low body weight gain (days 0-91) that was considered a compound-related effect. After the first three months, this group showed an apparent recovery, illustrated by body weight gains that were similar to those in other treated groups. Over the duration of the first year, body weight gains for the 20, 125, and 750 ppm groups of females were lower than female controls, but these differences could not be directly attributed to the dietary intake of mancozeb.

There were no compound-related differences in mean daily food consumption or mean food efficiency. In the male groups fed diets that contained 0, 20, 60, 125, or 750 ppm mancozeb, mean daily intake during the study was 0, 0.769, 2.33, 4.83, and 30.9 mg mancozeb/kg body weight/day, respectively. Mean daily intake by female rats in the same respective dietary concentration groups was 0, 1.06, 3.06, 6.72, and 40.2 mg mancozeb/kg body weight/day.

A low incidence of diarrhea among male rats fed 750 ppm mancozeb diets was the only clinical sign attributed to compound intake in rats in this study. None of the lesions observed during the ophthalmological examination of rats at the end of the study were considered compound related.

Early deaths among rats in the study were not attributed to compound-related effects. In general, male survival rates were lower than those for female rats. The percent survival for male rats fed 0, 20, 60, 125, or 750 ppm mancozeb in study diets was 26%, 29%, 39%, 16%, and 21%, respectively, and for female rats fed the same dietary levels was 37%, 53%, 39%, 39%, and 42%.

Clinical laboratory evaluations that included hematology, clinical chemistry, and urinalyses showed no compound-related effects in rats during the study. Two parameters that measure thyroid function were statistically different from controls in both male and female rats fed 750 ppm diets. Mean thyroxine (T₄) levels were lower than T₄ levels of controls and levels of thyroid stimulating hormone (TSH) were higher in these groups than in respective male and female control groups.

At the 12-month sacrifice, statistically significant elevated mean relative liver and testes weights were seen in male rats fed 750 ppm diets. The mean absolute weights of these organs were similar to the mean liver and testes weights from the male controls; therefore, the increased relative weights were attributed to the lower whole body weights of male rats in this dietary concentration group. None of the gross pathology observations during the 12-month necropsy were statistically significant; however, several significant microscopic observations were noted. Follicular cell hypertrophy/hyperplasia of the thyroid gland was visible in male and female rats fed diets that contained 750 ppm mancozeb. Changes appeared more severe in males than females. No thyroid changes were observed in rats fed dietary concentrations of 125 ppm or lower. Granular pigment was visible microscopically in the kidneys of male and female rats fed 125 ppm or 750 ppm diets, but no tissue alteration was associated with the occurrence of this pigment. At the 24-month sacrifice and necropsy, male rats fed 750 ppm diets had an elevated mean relative liver weight, and male and female rats fed these diets had elevated mean absolute and relative thyroid/parathyroid weights. The high relative liver weight was considered due to the low mean body weight for this group. The elevated thyroid weights were attributed to intake of mancozeb.

Gross changes observed during the necropsy of rats during the second year of the study included enlarged thyroids in both males and females fed 750 ppm diets and thyroid masses in the male rats in this dietary group.

A number of compound-related histopathological changes were observed in tissues from rats that died or were sacrificed during the second year of the study. Male and female rats fed 750 ppm diets had increased incidences of thyroid follicular cell carcinomas, adenomas, nodular hyperplasia and hypertrophy/hyperplasia. The incidences of rats with carcinomas or adenomas were higher for males (14/61 and 20/61, respectively) than for females (4/61 and 6/61). These thyroid changes are considered to be related to ethylene thiourea (ETU), a known thyroid carcinogen and product of mancozeb metabolism in the rat. ETU belongs to a class of compounds that inhibits synthesis of thyroid hormone and induces release of high TSH levels by the pituitary. Thyroid follicular cell adenomas and carcinomas are believed to result when the threshold for the thyroid-pituitary feedback mechanism is exceeded.

Changes were also evident during microscopic evaluation of the eyes of rats fed 750 ppm diets. Increased incidences and severity of bilateral retinopathy were noted for both male and female rats. The yellow-brown pigment in the kidney, noted during examination of the rats from the 12-month sacrifice, was also noted in rats in the 125 and 750 ppm dietary groups during the second year of the study. No tissue pathology was associated with the pigment. Although the presence of this pigment appeared to result from the intake of test material, it was determined not to have been related to any adverse biological effect.

The notable toxic effects attributed to the dietary intake of test material for the study were seen in rats fed diets that contained 750 ppm mancozeb. These effects included low body weight gains, alterations in thyroid hormone levels, increased thyroid weights, thyroid follicular cell hypertrophy/hyperplasia, thyroid adenomas, thyroid carcinomas, and bilateral retinopathy. The no-observable-adverse-effect level for rats fed diets that contained mancozeb during the study was 125 ppm (which corresponds to 4.8 mg mancozeb/kg bw/d in males and 6.7 mg mancozeb/kg bw/d in females.