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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-08-12 to 1986-08-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Please refer to the field 'Principles of method if other than guideline'.

Principles of method if other than guideline:

The text below is taken from the Draft (Renewal) Assessment Report prepared according to the Commission Regulation (EU) N° 1107/2009 of Mancozeb, Volume 3 - B.6 (AS).
Deviations from current OECD 416 (2001)
-Organ weight measurements were not performed for uterus, epididymides, prostate, seminal vesicles, brain, spleen pituitary and adrenal glands.
- oestrus cycle and sperm parameters were not investigated;
- sexual maturation parameters were not measured in F1 offspring.
It is considered that these limitations do not compromise the validity of the study as there is a wealth of regulatory and published repeated dose toxicity and reproductive toxicity data, including DNT studies in multiple species and mechanistic information showing that mancozeb does not affect the reproductive organs, sex hormones and related parameters.

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 animals

Species:

rat

Strain:

other: Crl: CD BR SD rats

Details on species / strain selection:

The strain was selected because background control data are available from previous studies conducted at Rohm and Haas Company.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Lakeview Facility, Nutley, NJ, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 21 days
- Weight at study initiation: (P) 35-50 g
- Housing: individually in a stainless steel cage suspended above an absorbent paper liner that was changed three times/week
- Diet: Certified Purina Rodent Chow #5001M ad libitum
- Water: ad libitum, available via an automatic watering system,
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
A pre-mix was prepared by weighing the appropriate amount of mancozeb for each dietary concentration and blending it with approximately 1 kg of certified rodent meal for 15 minutes in a Hobart N-50 planetary mixer. The pre-mix and the balance of untreated feed were added to a Patterson-Kelly cross-flow blender and blended for an additional 15 minutes to prepare the final diets. The control diet was prepared in the same manner as the treated diets. Fresh diets were prepared weekly and fed ad libitum. Uneaten or unused diet was discarded as hazardous waste.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 10 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in polycarbonate cages containing Alpha-Dri® bedding

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

When the first batch of diets were prepared, samples from the top, middle, and bottom of each dietary concentration were collected (2/area/dose) and submitted for analysis of active ingredient to determine uniformity of the blend. Each week, an extra feed cup was prepared for each dietary concentration and left on top of a cage bank in the study room during the treatment week. At the end of the week, these retention samples (ret samples) were collected and 14 representative samples from each dose level were submitted for analysis of active ingredient to verify compound stability during the feeding interval.

Duration of treatment / exposure:

The P1 generation (males and females) was offered diets containing mancozeb continuously from 42 days of age throughout the pre-mating period (minimum of 10 weeks), and throughout the mating, gestation and the lactation periods. The P2 generation (males and females) was exposed to mancozeb from conception through weaning. After weaning, the P2 animals were offered diets containing mancozeb for a minimum of 10 weeks (pre-mating period) and throughout the mating, gestation and lactation periods. Males were killed after the second mating period; females, after the F2b litter was weaned.

Frequency of treatment:

daily

Details on study schedule:

When the youngest litter reached 25 days of age, one male and one female from each litter was selected randomly to serve as parents (P2) for the F2 generation. If a group did not have 25 pairs of animals, then a second male and female was selected randomly from the remaining litters (no more than 1/sex/litter) to make up the required number of pairs. The week in which the P2 generation was selected was designated Week 1 for purposes of measuring body weight, feed consumption and clinical signs. Cage site checks were conducted twice a day (once a day on weekends and holidays) during the post-weaning period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

0 mg/kg bw/d

Doses / concentrations 2

Dose / conc.:

30 ppm

Remarks:

Compound intake (mg/kg bw/d):

Males
P1 Pre-mating: 1.73 ± 0.23

Females
P1 Pre-mating 2.06 ± 0.41
P1 / F1a Gestation 2.03 ± 0.06
P1 / F1a Lactation 4.00 ± 1.21
P1 / F1b Gestation 1.83 0.12
P1 / F1b Lactation 3.70 ± 1.50

Males
P2 Pre-mating 2.11 ± 0.47

Females
P2 Pre-mating 2.49 ± 0.41
P2 / F2a Gestation 2.13 ± 0.06
P2 / F2a Lactation 4.23 ± 1.34
P2 / F2b Gestation 1.97 0.06
P2 / F2b Lactation 4.13 ± 1.26

Doses / concentrations 3

Dose / conc.:

120 ppm

Remarks:

Compound intake (mg/kg bw/d):

Males
P1 Pre-mating: 6.95 ± 1.01

Females
P1 Pre-mating 8.22 ± 1.57
P1 / F1a Gestation 8.27 ± 0.15
P1 / F1a Lactation 17.53 ± 5.80
P1 / F1b Gestation 7.47 ± 0.23
P1 / F1b Lactation 16.23 ± 7.00

Males
P2 Pre-mating 8.61 ± 2.07

Females
P2 Pre-mating 10.52 ± 1.70
P2 / F2a Gestation 8.60 ± 0.36
P2 / F2a Lactation 18.80 ± 6.56
P2 / F2b Gestation 7.90 ± 0.40
P2 / F2b Lactation 17.53 ± 5.52

Doses / concentrations 4

Dose / conc.:

1 200 ppm

Remarks:

Compound intake (mg/kg bw/d):

Males
P1 Pre-mating: 68.90 ± 10.19

Females
P1 Pre-mating 83.90 ± 18.03
P1 / F1a Gestation 86.67 ± 4.39
P1 / F1a Lactation 183.83 ± 54.02
P1 / F1b Gestation 79.37 ± 2.71
P1 / F1b Lactation 168.13 ± 53.94

Males
P2 Pre-mating 87.11 ± 20.09

Females
P2 Pre-mating 114.26 ± 16.36
P2 / F2a Gestation 93.20 ± 3.66
P2 / F2a Lactation 186.50 ± 51.89
P2 / F2b Gestation 84.37 ± 2.31
P2 / F2b Lactation 172.43 ± 49.96

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for clinical signs of reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly on all adult animals

FOOD CONSUMPTION AND COMPOUND INTAKE
Male feed consumption were recorded weekly until cohabitation. Female feed con sumption were taken weekly during the pre-mating period. Feed consumption of presumed pregnant females was recorded on Days 0, 7, 14, and 21 of gestation. Feed comsumption of females that produced viable litters was recorded on Days 0, 7, 14, and 21 of lactation.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 5/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after the last litters in each generation were produced
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of: All organs, tissues, and body cavities were examined in adult P1 and P2 animals found dead or those animals scheduled for necropsies.

HISTOPATHOLOGY / ORGAN WEIGHTS
Complete necropsies were performed on all P1 and P2 rats. Liver (weighed), thyroid (weighed post-fixation), pituitary, testes (weighed), epididymides, prostate, seminal vesicles, coagulating gland, ovaries (weighed), uterus, vagina, cervix, and gross lesions were collected from all animals. In addition, kidneys (weighed) were collected from P2 rats of both sexes and P1 female rats.
Microscopic examination of hematoxylin and eosin stained sections was performed on all tissues collected from control and 1200 ppm groups and gross lesions from the lower treatment groups of P1 and P2 rats. In addition, thyroid, pituitary, and kidney (females only) were examined from lower treatment groups of P1 animals. Thyroid, pituitary, kidney, and liver were examined from lower treatment groups of P2 animals.
Perls' Iron stain was performed on sections of kidney and/or liver from representative P2 male rats receiving 1200 ppm (4 animals) and from the control group (4 animals)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.

Statistics:

The litter (i.e., proportion of affected fetuses/litter or litter mean) was used as the experimental unit for the purpose of statistical evaluation. The level of significance selected was p<0.05. The statistical tests that were used to analyze the parameters studied are:
Fisher's exact test (Incidence of pregnancy, clinical signs, maternal death, litters with stillborn pups, gross necropsy, histopathology)
Mann-Whitney U test (live and dead fetuses/litter, sex ratio)
Dunnett's test (parental body weight and feed consumption, offspring body weight, absolute and relative organ weights, length of gestation)

Reproductive indices:

Mating Index =(Number of females that mated/Number of females used for mating)*100;

Fertility Index = (Number pregnant females/Number of females mated)*100;

Gestation Index= (Number of females producing litters with at least one live pup/Number of pregnant females)*100;

Lactation Index= (Total number of pups alive on Day 4 PP/Number of pups bom alive)*100

Offspring viability indices:

Viability Index=(Total number of pups alive at weaning/Number of pups alive after culling (Day 4 PP))*100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One P1 dam in the 30 ppm group died on Day 19 of gestation and one P1 dam in the 120 ppm group died while delivering during the second mating period. These deaths were not considered treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1200 ppm, the mean body weight of P1 male rats was significantly less than the control group by the second week of treatment. Their body weights remained significantly below the control value throughout the pre-mating period. The mean body weight of P1 female rats at 1200 ppm was significantly less than the control group by the third week of treatment and remained depressed throughout the pre-mating period. The effect on male and female body weight at 1200 ppm was considered treatment-related. Mean body weight for P1 females at 1200 ppm continued to be depressed throughout the gestation and lactation periods for the F1a and F1b generations. The effect on body weight was considered treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean feed consumption at 1200 ppm was decreased among P1 male and female rats throughout the 10 weeks of treatment prior to mating. This effect on feed consumption at 1200 ppm was considered treatment-related. At 1200 ppm mean feed consumption of P1 females was slightly lower than the control value during gestation for the F1a generation. Mean feed consumption of the P1 females returned to the control value during the lactation period for the F1a generation and remained at control levels throughout the gestation and lactation periods for the F1b generation. A random decrease in feed consumption was noted among dams at 30 ppm during the first week of lactation for the F1a generation. The decrease was not considered treatment-related.

Food efficiency:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic changes were observed in the thyroid, kidney, and pituitary. Diffuse and nodular hyperplasia of the thyroid follicular cells in both sexes, and follicular adenomas in the males were treatment-related in rats at 1200 ppm. Also noted at 1200 ppm was an increase in the severity of hypertrophy and/or vacuolation of individual cells of the anterior pituitary in males.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Follicular adenomas in the males were treatment-related in rats at 1200 ppm

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

P1Adult/F1a Offspring:
The number of litters with one or more stillborn pups was slightly higher at 120 and 1200 ppm. The differences were not statistically significant and were not considered a result of treatment. The total number of stillborn offspring was higher in the 30, 120 and 1200 ppm group when compared to the control value. The difference was not considered treatment-related since the majority of stillborn offspring were contributed by one litter in each of the treated groups (all of the seven stillborn found in one litter at 30 ppm; five of the nine stillborn found in one litter at 120 ppm; three of the six stillborn found in one litter at 1200 ppm). In addition, the number of stillborn observed did not appear dose-dependent. There were no significant increases between the control group and the treated groups in the number of offspring dying during lactation. The viability and lactation indices were similar in the control group and treated groups. There were no treatment-related effects on mean number of live pups/litter on Days 0, 4, 7, 14, and 21 PP. A statistically significant decrease in the number of live pups/litter on Days 14 and 21 of lactation at 3 0 ppm was artificial and not related to treatment. Six of 22 litters at 30 ppm had eight or less offspring. These litters reduced the mean number of offspring/litter after culling to nine instead of the 10 offspring/litter anticipated.
P1 Adults/F1b Offspring: no effects

Details on results (P0)

Gross pathological findings in the P1 generation
There were no treatment-related gross observations that could be substantiated by corresponding microscopic findings.
There was a statistically significant increased incidence of prominent architecture of the liver in females at 1200 ppm compared to controls. This observation was made for 2/25 control and 6/25, 6/25, and 9/25 female rats at 30, 120, and 1200 ppm, respectively. Prominent lobular architecture was observed with comparable incidence in male rats from the control and 12 00 ppm group. No microscopic change could be consistently correlated with this observation.
Flaccid and/or small testes were observed grossly in 1/25, 1/25 and 2/25 rats at 30, 120, and 1200 ppm, respectively, but not in controls. In all cases, this change was unilateral. In the rats at 120 and 1200 ppm this observation was correlated microscopically with unilateral atrophy; the rat at 30 ppm with this observation was normal. The incidence of testicular atrophy (unilateral or bilateral) observed microscopically was 3/25 in controls and 5/25 in rats at 12 00 ppm.
Enlargement of the uterus, which was generally slight, was observed in 2/25, 1/25, and 4/25 rats at 30, 120, and 1200 ppm, respectively, but not in controls. This observation generally corresponded to dilation microscopically, which was comparable between controls and rats at 1200 ppm.
Treatment-related microscopic changes were observed in the thyroid, kidney, and pituitary.
A spectrum of changes involving the follicular cells of the thyroid was observed. A statiscally significant incidence of diffuse hyperplasia occurred in 25/25 male and 22/25 female rats at 1200 ppm. Diffuse hyperplasia was also observed in one female at 120 ppm. This change was characterized by enlarged, often angularly shaped follicles lined by moderately tall follicular cells which sometimes formed papillary infoldings. Diffuse hyperplasia was minimal to moderate in males and minimal to mild in females at 1200 ppm. This change was minimal in the female at 120 ppm. In addition, well-circumscribed focal or nodular areas of hyperplasia, which were sometimes cystic, were observed in two males at 1200 ppm. One male at 120 ppm had a focus of nodular/cystic follicular cell hyperplasia without the diffuse change. Follicular cell adenoma was observed in three males at 1200 ppm.
The minimal diffuse hyperplasia observed in one female at 12 0 ppm is not considered to be treatment-related since this change was observed in control P2 rats. Nodular/cystic follicular cell hyperplasia which was observed in one male at 120 ppm may represent a spurious occurrence.
Brown globular pigment was observed within the lumen of proximal tubules in the kidneys at statistically significant incidences in rats of both sexes at 120 and 1200 ppm. This pigment was limited to the luminal space with no associated changes to the tubular epithelium. With histochemical stains performed on kidneys containing similar pigment from a three-month dietary toxicity study of Dithane M-4 5®in rats, this pigment was negative with the iron, acid fast and bile stains and positive with the PAS stain (7,8). A minimal to mild amount of pigment was observed in the kidneys of all nine male rats at 1200 ppm that were collected because of gross observations. Females at 1200 ppm had minimal to moderate amounts of pigment in all 25 rats examined. Four of six male rats that had kidneys collected because of gross observations and 19/25 female rats at 120 ppm had trace to minimal amounts of pigment.
Hypertrophy and/or vacuolation of individual cells in the adenohypophysis (anterior or glandular portion) of the pituitary was observed in 10/24, 20/25, 18/25, and 20/25 males and 1/25, 9/24, 11/24 and 8/25 females from the control, 30, 120, and 1200 ppm groups, respectively. This finding, always minimal in females and minimal or mild in males from the control, 30 ppm, and 12 0 ppm groups, was moderate in three males at 12 00 ppm. The increased incidence observed in treated groups (only statistically significant in females) did not occur in treated rats from the P2 generation. Therefore, hypertrophy and/or vacuolation was considered to be treatment-related only in males at 1200 ppm due only to the somewhat increased severity.
Other microscopic changes occurred with comparable incidences between groups or occurred sporadically and were not considered to be related to treatment. An adenoma of C-cells in the thyroid was observed in one male and female each at 1200 ppm. In addition, mild focal hyperplasia of C-cells occurred in one male at 1200 ppm and one female at 120 ppm. These findings were not considered to be related to treatment. This conclusion was supported by the fact that minimal focal C-cell hyperplasia alone occurred in the P2 generation in one female rat each from the control, 30 ppm and 120 ppm groups.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

70 mg/kg bw/day

Based on:

other: lowest compound intake in the 1200 ppm dose group

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

histopathology: neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

7 mg/kg bw/day

Based on:

other: lowest compound inake in the 120 ppm dose group

Sex:

male/female

Basis for effect level:

other: no effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 200 ppm

System:

other: endocrine and urinary system

Organ:

kidney

pituitary gland

thyroid gland

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In P2 animals at 1200 ppm, mean body weight among male and female rats was significantly below control values throughout the 10 weeks of treatment prior to mating. The effect on mean body weight at 1200 ppm was considered a result of treatment. Mean body weight of P2 females at 1200 ppm remained below the control values throughout the gestation and lactation periods for the F2a and F2b generation. This effect on body weight at 1200 ppm was considered treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The increase in relative liver weight noted among males at 120 ppm is being considered treatment-related. However, this change is equivocal since the magnitude of the change was slight (5%), the effect was not noted among P2 males or P2 females, the absolute liver weight was similar to the control value, and no histopathologic changes were evident. Treatment-related increases were noted in relative liver weights and absolute and relative thyroid weights among males and females at 1200 ppm. Females at 1200 ppm also showed treatment-related increases in relative kidney weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related gross observations that could be substantiated by corresponding microscopic findings. For details, please refer to the field 'Details on results'.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic changes were observed in the thyroid, kidney, and pituitary. Diffuse and nodular hyperplasia of the thyroid follicular cells in both sexes, and follicular adenomas in the males were treatment-related in rats at 1200 ppm, with generally higher incidences or severity in the second generation compared to P1. Also noted at 1200 ppm was an increase in the severity of hypertrophy and/or vacuolation of individual cells of the anterior pituitary in males.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Follicular adenomas in the males were treatment-related in rats at 1200 ppm, higher incidence than in P1.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The increase in the total number of stillborn pups at 1200 ppm resulted from one litter. When the litter was observed on Day 0 PP seven pups were dead (presumed stillborn) and two were cannibalized. This result was not considered treatment-related. An increase in offspring death at 30 ppm between Days 8-14 PP was due to one litter that contributed three of the six dead pups for that time period. The increase in offspring death at 1200 ppm between Days 8-14 PP was due to a litter that contributed 10 (cannibalized) of the 11 dead pups for that time period. The increases at 30 and 1200 ppm were not considered treatment-related. A slight decrease in pup survival on Day 21 PP at 30 and 1200 ppm appeared to be due to the increased offspring death between Days 8-14 PP and due to the unusually high survival rate in the control group (99%) at Day 21 PP.
P2 Adults/F2b Offspring:
A statistically significant increase in the total number of male pups at 30 ppm on Day 0 PP appeared to be due to the unusually low number of males in the control group and not related to treatment.

Details on results (P1)

Gross Pathological Findings in the P2 animals

There were no treatment-related gross observations that could be substantiated by corresponding microscopic findings.
There was a statistically significant increased incidence of prominent lobular architecture of the liver in males at 1200 ppm compared to controls. This observation was made for 7/25 control and 7/25, 8/25 and 18/25 male rats at 30, 120 and 1200 ppm, respectively. Prominent lobular architecture was observed with comparable incidence in female rats from the control and 1200 ppm groups. No microscopic change could be consistently correlated with this observation.
Flaccid and small testes were observed grossly in 1/25 rats at 30 ppm, in which it was bilateral, and 1/25 rats at 1200 ppm in which it was unilateral. Microscopically, both of these rats had bilateral atrophy. Two rats at 30 ppm had the observation of unilateral enlargement of the testes, which corresponded microscopically to unilateral atrophy of the opposite side. The incidence of testicular atrophy (unilateral or bilateral) observed microscopically was 3/25 in controls and 3/24 in rats at 1200 ppm.
Treatment-related microscopic changes were observed in the thyroid, kidney and pituitary.
The same spectrum of changes involving the follicular cells of the thyroid observed in the P± generation was also observed in the P2 generation with generally higher incidences or greater severity. A statistically significant incidence of diffuse hyperplasia occurred in 24/24 males and females each at 1200 ppm. This change was generally moderate in males and mild in females. In addition, minimal diffuse hyperplasia was observed in 2/25, 4/25 and 2/25 male rats from the control, 30 ppm, and 120 ppm groups, respectively. Nodular/cystic follicular cell hyperplasia occurred in 9/24 males (statistically significant) and 4/24 females at 1200 ppm. This change was observed in one male and female each at 30 ppm. Follicular cell adenoma was observed in four males at 1200 ppm, one of which had been observed grossly.
The minimal diffuse hyperplasia observed in male rats at 3 0 ppm and 120 ppm is not considered to be treatment-related since this change was also observed in controls with similar incidences and severity. The occurrence of nodular/cystic follicular cell hyperplasia in one male and female each at 30 ppm may represent spurious occurrences.
Pigment in the lumen of proximal tubules in the kidney was observed at statistically significant incidences in all rats at 1200 ppm with generally mild amounts in males and moderate amounts in females. Trace to minimal amounts of pigment were present in 19/25 male and 11/25 female rats at 120 ppm (statistically significant). One male at 30 ppm with dilated tubules, regenerative tubular epithelium and interstitial mononuclear cells indicative of advanced nephropathy sufficient to interfere with renal function, had minimal amounts of luminal pigment. Luminal pigment was negative for iron in representative sections of kidney from males at 1200 ppm stained by Perls' method.
Hypertrophy and/or vacuolation in the adenohypophysis of the pituitary was observed in 20/25, 19/25, 20/25, and 21/24 males and 9/2 5, 15/25, 9/25 and 16/24 females from the control, 30, 120 and 1200 ppm groups, respectively. This finding was always minimal in females and minimal to moderate in males. It was considered to be treatment-related only in males at 12 00 ppm due to the somewhat increased severity.
Other microscopic changes occurred with comparable incidences between groups or occurred sporadically and were not considered to be related to treatment.

Effect levels (P1)

1

Dose descriptor:

LOAEL

Effect level:

70 mg/kg bw/day

Based on:

other: lowest compound intake in the 1200 ppm dose group

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

histopathology: neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

7 mg/kg bw/day

Based on:

other: lowest compound intake in the 120 ppm dose group

Sex:

male/female

Basis for effect level:

other: no effects

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 200 ppm

System:

other: endocrine and urinary system

Organ:

kidney

pituitary gland

thyroid gland

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no significant increases between the control group and the treated groups in the number of F1 offspring dying during lactation. The viability and lactation indices were similar in the control group and treated groups. There were no treatment-related effects on mean number of live pups/litter on Days 0, 4, 7, 14, and 21 PP. A statistically significant decrease in the number of live pups/litter on Days 14 and 21 of lactation at 3 0 ppm was artificial and not related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A treatment-related decrease in mean litter weight was noted on Day 21 PP among offspring at 1200 ppm and may be a result of older offspring eating treated diet late in the lactation period since mean body weights from this group were unaffected while the offspring were nursing.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Sexual maturation:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

70 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Remarks on result:

other: 70 mg/kg bw/d based on the lowest compound intake, 1200 ppm dose group

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

When the F2a litter was observed on Day 0 PP seven pups were dead (presumed stillborn) and two were cannibalized. This result was not considered treatment-related. There were no treatment-related effects on the total number of offspring dying during the lactation period or in the corresponding viability or lactation indices. An increase in offspring death at 30 ppm between Days 8-14

Body weight and weight changes:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

70 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Remarks on result:

other: 70 mg/kg bw/d based on the lowest compound intake, 1200 ppm dose group

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

As no adverse effects on reproduction were noted, the NOAEL for reproductive effects and offspring toxicity is determined to be 1200 ppm (70 mg/kg bw/d), the NOAEL for parental toxicity was 120 ppm (7 mg/kg bw/d). Both values in mg/kg bw/d were calculated based on the lowest compound intake (dietary administration).

Executive summary:

Mancozeb (puritiy 83.3 % a.i.) was administered in the diet through two generations of rats with two mating periods per generation. Mancozeb was administered in the diet to four groups (25 rats/group/sex) at concentrations of 0 (control), 30, 120 and 1200 ppm (of active ingredient) in both the first parental (P1) and the second parental (P2) generations. The P1 generation (males and females) was offered diets containing mancozeb continuously from 42 days of age throughout the pre-mating period (minimum of 10 weeks), and throughout the mating, gestation and the lactation periods. The P2 generation (males and females) was exposed to mancozeb from conception through weaning. After weaning, the P2 animals were offered diets containing mancozeb for a minimum of 10 weeks (pre-mating period) and throughout the mating, gestation and lactation periods. Control rats were fed untreated diets through the same periods. Adult animals were observed at least once daily for signs of ill health or reaction to treatment. Male body weight and feed consumption were recorded weekly during the P1 and P2 pre-mating period. Female body weight and feed consumption were recorded weekly during the pre-mating period, the gestation and lactation periods. Physical exams were performed weekly on all adult animals. Offspring were observed at least once daily to detect dead or moribund animals. On Days 4, 7, 14 and 21 postpartum (PP) the offspring were individually handled, weighed and examined for abnormal behaviour or appearance. Necropsies were performed on all P1 and P2 adults (males, after the second mating period; females, after their second litter was weaned). The reproductive organs, liver, thyroid, P1tuitary, kidneys and any gross lesions were collected and prepared for histopathologic examination for all P1 and P2 adult rats. The liver, kidneys, thyroid and testes/ovaries weights were recorded at necropsy.

No treatment-related deaths or signs of ill health or reaction to treatment occurred in either the P1 or P2 adults. Mean body weight of male and female rats (P1 and P2 generations) were similar in the control group and 30 and 120 ppm groups throughout the study. At 1200 ppm, treatment-related decreases in mean body weight occurred in male and female rats (P1 and P2 generations) throughout the pre-mating period. Mean maternal body weight among P1 and P2 females at 1200 ppm remained depressed throughout the gestation and lactation periods. Mean feed consumption of male and female rats (P1 and P2 generations) were similar in the control group and the 30 and 120 ppm groups throughout the study. A significant decrease in mean feed consumption was evident at 1200 ppm among P1 male and females rats throughout the pre-mating period and among P1 female rats throughout the F1 gestation period. No treatment-related effects were noted for the P2 male and female rats at 1200 ppm during the pre-mating, gestation or lactation periods.

No effect on reproductive function was noted after the first or second mating of the P1 or P2 adults. Indices for mating, fertility, gestation, viability and lactation were similar in the control group and treated groups. Mean body weight of the offspring was similar between the control group and treated groups in the F1a, F2a and F2b generations. A treatment-related decrease in offspring body weight was noted at Day 21 PP in the F1b generation at 1200 ppm. Sex ratios were similar between the control and treated groups in all generations. An increase was noted in the relative liver weight of P2 males at 120 ppm. Significant increases in both relative liver weight and absolute and relative thyroid weights occurred in both P1 male and female rats and in relative kidney weight among females at 1200 ppm. Treatment-related increases were noted in relative liver weights and absolute and relative thyroid weights among P2 males and females at 1200 ppm. Relative kidney weights were also increased in P2 females at 1200 ppm.

There were no treatment-related gross changes that could be substantiated by corresponding microscoP1c findings among adult male and female rats in the P1 or P2 generations. No treatment-related microscopic changes occurred in reproductive organs of either sex. Treatment-related microscopic changes occurred in the thyroid, kidney and P1tuitary of both the P1 and P2 generations. Diffuse hyperplasia of follicular cells, nodular/cystic follicular cell hyperplasia, and follicular cell adenoma were treatment-related in the thyroid of rats of both sexes at 1200 ppm with higher incidences or severity in P2 rats. An increase in the severity of hypertrophy and/or vacuolation of individual cells in the adenohypophysis of the pituitary occurred in males only at 1200 ppm in both P^ and P2 animals. Brown globular P1gment was observed within the lumen of proximal tubules in the kidneys of rats of both sexes at 120 and 12 00 ppm with generally similar incidence and severity in both generations.

 

Conclusion

Mancozeb when administered in the diet to rats for two generations at concentrations of 0 (control), 30, 120 and 1200 ppm produced no adverse effects on reproductive capability or on the health and survival of offspring. In the parental animals no treatment-related effects were seen at 30 ppm. The only effects seen at 120 ppm were an increase in relative liver weight among P2 males and an increased incidence of pigment in the proximal tubules of the kidney. The toxicologic significance of these findings are equivocal.