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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on fertility were noted in the available 2-Generation study in rats.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

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

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

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.

Dose / conc.:

0 ppm

Remarks:

0 mg/kg bw/d

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

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

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

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

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: 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

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.

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

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

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

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: 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.

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.

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

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

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and Guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity was determined to be 128 mg/kg bw/d.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-06-14 to 2015-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

Details on mating procedure:

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

Duration of treatment / exposure:

14 days (GD 6 - 19 )

Frequency of treatment:

daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

vehicle control

No. of animals per sex per dose:

25 / pregnant females / dose

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

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

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

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

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

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

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

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

Ovaries and uterine content:

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

Blood sampling:

not examined

Fetal examinations:

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

Statistics:

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

Indices:

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

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

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

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

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

Clinical signs:

no effects observed

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

40

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

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

Skeletal malformations:

no effects observed

Description (incidence and severity):

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

Visceral malformations:

no effects observed

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

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

Executive summary:

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

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

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

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986-01-13 to 1986-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

The duration of treatment did not cover the period from implantation until end of gestation, but only the organogenesis period. It is considered that this limitation does not compromise the validity of the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Devaiations from current OECD 414 (2001):
- The duration of treatment did not cover the period from implantation until end of gestation, but only the organogenesis period. It is considered that this limitation does not compromise the validity of the study as there are more modern developmental toxicity studies and a DNT study in rats in which animals were exposed before organogenesis. Although there are no modern developmental toxicity studies in rabbits, mancozeb is not a developmental toxicant in rabbits and it is unlikely that exposure before organogenesis would affect this conclusion (please refer to Draft (Renewal) Assessment Report prepared according to the Commission Regulation (EU) N° 1107/2009 of Mancozeb, Volume 3-B.6)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Female rabbits:
- Source: Hazleton Research Animals, Denver, PA, USA
- Age at study initiation: 4 to 5 month old
- Weight at study initiation: 3.2-3.8 kg
- Diet: about 225 g daily of Purina certified High Fiber Rabbit Chow #5325
- Water: ad libitum via filtered water system
- Acclimation period: 41 days
Housing: individually in a stainless steel cage suspended above absorbent paper liners that were changed three times/week. Nesting materials were not provided because the does were killed the day before expected delivery.

Male rabbits: The stock male rabbits used in this study were of the same strain and obtained from the same supplier and are maintained at the Rohm and Haas Toxicology Department specifically for breeding purposes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test material in 0.5% methylcellulose were prepared daily and administered by gavage on Days 7-19 of gestation at a volume of 5 mL/kg of body weight. For each dosing suspension, mancozeb was weighed and added to a 500 mL volumetric flask containing about 50 mL of the methylcellulose solution. Additional methylcellulose was then added to the flask to make 500 mL of the test suspension. Once prepared, the test suspensions were stirred continuously to ensure homogeneity. All dose suspensions were administered within 30 minutes of preparation. Total dose administered each day was based on the most recently recorded body weight of each animal. The vehicle control group was given 0.5% methylcellulose only, using the same route, volume, and dosing schedule.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test suspensions were taken on the second, ninth, and last days of the dosing period. Test samples (1 mL each) from each dose level were taken and frozen immediately. Frozen test suspension samples were submitted to the Analytical Chemistry Department, Rohm and Haas Company for analysis of active ingredient.

Details on mating procedure:

- Impregnation procedure: artificial insemination
- The day of inseminating was designated as Day 0 of gestation.

Duration of treatment / exposure:

days 7 to 19 of gestation

Frequency of treatment:

daily

Duration of test:

Females were killed on gestation day 29.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment:
Eighty females selected for the study were divided into four lots of 20 animals each. The four lots were inseminated on consecutive days. Each day, the inseminated females were randomly assigned to the control or treated groups (5/group/day) using a table of random numbers. After assigning all females to groups, the mean Day 0 G body weight per group was calculated and statistically analyzed to insure that no significant differences existed among the groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Morbidity and mortality checks were performed in the afternoon on Days 0-6 and Days 20-28 G.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were taken in the morning of Days 0-29 G and in the afternoon of Days 7-19 G (dosing period).

BODY WEIGHT: Yes
- Time schedule for examinations: The females were weighed on Days 0, 7, 9, 11, 14, 17, 20 and 29 G.

FOOD CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: During the necropsy animals were examined for evidence of pregnancy and gross lesions of the abdominal and thoracic cavities.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter

Statistics:

The litter (the proportion of affected fetuses/litter or the litter mean) was considered the experimental unit for the purpose of statistical evaluation. The level of significance selected was p<0.05. A pair-wise test between the control and treated groups was applied to each parameter.

Indices:

No indices calculated. The number of abortions, litters produced, and the mean number per litter or corpora lutea as well as implantations, resorptions, dead and live foetuses and sex ratio were recorded.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Significant increases in the incidence of alopecia, anorexia and scant feces were noted at 80 mg/kg bw/d during the treatment period. At 80 mg/kg bw/d, an increased incidence of anuria, no feces, ataxia, red discharge on the cageliner and stained perineum noted between Days 7-19 G were considered to be treatment related even though they were not significantly different from the control value. During the post-treatment period (Days 20-29 G) significant increases were noted in anorexia, scant or no feces, and red discharge on the cageliner at 80 mg/kg bw/d. During the post-treatment period, increases in alopecia and ataxia among does at 80 mg/kg bw/dwere considered to be treatment-related even though they were not statistically significant from the control value.

Mortality:

mortality observed, treatment-related

Description (incidence):

No treatment-related deaths occurred in the control group or in the 10 and 30 mg/kg bw/dgroups. Two maternal deaths occurred at 80 mg/kg bw/d. Both deaths were considered treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Adverse effects on body weight were noted among the five does that aborted at 80 mg/kg bw/d. During the treatment period, losses in body weight among these does ranging from 210g to 720g, were considered treatment related. Body weight gain among does at 8 0 mg/kg bw/d that produced at least one viable fetus were similar to the control value.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption was severely reduced among the five does that aborted in the 80 mg/kg bw/d group. This adverse effect was considered to be treatment-related. Feed consumption among does in the 80 mg/kg bw/d group that produced at least one viable fetus was similar to the control value.

Food efficiency:

not examined

Gross pathological findings:

no effects observed

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

At 80 mg/kg bw/d, a significant increase in does aborting (5/15) was reported with a corresponding decrease in the number of litters produced.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Dose descriptor:

LOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

other: decreased number of litters produced (due to abortion of fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: absence of effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Mean foetal body weight was similar between the control and treated groups. There were no significant increases in the types or incidence of malformations or developmental variations between control or treated groups.

Key result

Dose descriptor:

NOAEL

Effect level:

> 80 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the maternal NOAEL of mancozeb administered by gavage to New Zealand White rabbits during gestation days 7 to 19 was 30 mg/kg bw/d, the NOAEL for embryo/foetotoxic effects was > 80 mg/kg bw/d.

Executive summary:

Mancozeb was suspended in a 0.5% aqueous solution of methylcellulose and administered orally by gavage to 4 groups of artificially inseminated New Zealand white rabbits (20/group) at doses of 0 (control), 10, 30, and 80 mg/kg on Days 7-19 of gestation (G). All doses were administered at a constant volume of 5 mL/kg bw/d. The rabbits were observed daily for clinical effects and weighed with measurement of food consumption on days 0, 7, 9, 11, 14, 17, 20, and 29. On day 29 of gestation the does were killed and the thoracic and abdominal cavities were examined for gross pathological changes. The gravid and empty uterus was weighed; uterine contents and genital tract were examined for corpora lutea, implantation sites, and resorptions were counted. The number of live and dead foetuses per litter was counted with location in uterus recorded. Each foetus was weighed, sexed, and examined for external, visceral, and skeletal alterations.

No treatment-related deaths occurred in control or in the 10 and 30 mg/kg bw/d dose groups. One animal died in the 30 mg/kg bw/d group but death was attributed to mis-dosing. Two does were sacrificed in a moribund condition at the high dose and the deaths considered treatment-related (one of these does was pregnant and did not abort). Clinical signs were comparable between controls and animals receiving 10 and 30 mg/kg bw/d. Animals receiving 80 mg/kg bw/day manifested significant increases in clinical signs. Alopecia. anorexia, ataxia, scant faeces and abortions were all observed at the highest dose tested. Body-weight gain and food consumption were not significantly different between controls and groups receiving 10 or 30 mg/kg bw/d. At 80 mg/kg bw/d, body-weight and food consumption were significantly decreased in does that aborted and those sacrificed moribund. Does producing at least one viable foetus had body-weight gains and food consumption values similar to controls.

No treatment-related changes were evident in the incidence of gross post-mortem findings between does in the control and treated group. Reproductive parameters between controls and does receiving 10 or 30 mg/kg bw/d were comparable as measured by the number of abortions, litters produced, and the mean number per litter or corpora lutea, implantations, resorptions, dead and live foetuses or sex ratio. At 80 mg/kg bw/d, a significant increase in does aborting (5/15) was reported with a corresponding decrease in the number of litters produced. All other parameters were comparable to the control group. Mean foetal body weight was similar between the control and treated groups. There were no significant increases in the types or incidence of malformations or developmental variations between control or treated groups. The NOAEL for maternal toxicity was 30 mg/kg bw/d. The NOAEL for embryo/foetotoxicity was greater than 80 mg/kg bw/day. Maternal toxicity at 80 mg/kg bw/day was based on an increase in aborted foetuses, decreased number of litters produced, decreased body-weight gain and food consumption and an increase in clinical signs and death. Under the conditions of this study, the maternal NOAEL of mancozeb (83% pure) administered by gavage to New Zealand White rabbits during gestation days 7 to 19 was 30 mg/kg bw/d, based on an increase in aborted foetuses, decreased number of litters produced, decreased body-weight gain and food consumption and an increase in clinical signs and death at 80 mg/kg bw/d. The NOAEL for embryo/foetotoxic effects was equal to or greater than 80 mg/kg bw/d, the highest dose tested. It should be noted that the top dose of 80 mg/kg bw/d exceeds the MTD in rabbit maternal animals.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14.01.1980-26.01.1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: BLU: (SD) BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 13 weeks at mating
- Housing: individually
- Diet: Purina Laboratory Rodent Chow, ad libitum
- Water: fresh tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-3
- Humidity (%): ca 50

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

The positive control substance ETU was dissolved in distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared fresh daily and administered as a suspension in Mazola Corn Oil. The amount of test or control substance given to each animal was determined using the body weight on day 6 of gestation. This dose was maintained through day 15 of gestation.
The vehicle control and positive control animals received 100 percent corn oil and ETU in distilled water, respectively at an equivalent volume as the test animals.
Dose volume: 10 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Sexually mature females, proximately 13 weeks of age (weighing an average of 225 g) were mated 3:1 with sexually mature males. There were a sufficient number of matings to produce a total of 180 pregnant rats. The matings were managed by Blue Spruce Farms, Inc., Altamont, New York in four stages:
January 7, 1980, a sufficient number of rats were mated to produce at least 45 pregnant rats
January 8, 1980, 45 more,
January 9, 1980, 45 more and
January 10, 1980, 45 more;
yielding a total of 180 pregnant rats. The dams were then delivered to the Booz, Allen & Hamilton Inc. FDS Life Sciences Laboratory on January 12, 1980 by Blue Spruce Farms, Inc., Altamont, New York.

- M/F ratio per cage: 1/3
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

days 6 to 15 of gesation

Frequency of treatment:

daily

Duration of test:

On day 20 of gestation, all animals were sacrificed

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

2 mg/kg bw/day (nominal)

Dose / conc.:

8 mg/kg bw/day (nominal)

Dose / conc.:

32 mg/kg bw/day (nominal)

Dose / conc.:

128 mg/kg bw/day (nominal)

Dose / conc.:

512 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26 females

Control animals:

yes, concurrent vehicle

other: 26 females received the positive control ETU (50 mg/kg bw/d)

Details on study design:

- Rationale for animal assignment:
Each of the four groups of dams were distributed into the same seven treatment groups using a random number assignment sheet. Five test groups, a vehicle control group and a positive control group (each consisting of at least 26 animals) were established. Twenty-six animals were assigned to each group to allow for false pregnancies and maternal deaths. The randomization was conducted out of a table of random numbers from A Million Random Digits with 100,000 Normal Deviates, Rand Corporation, 1955. The first six or seven cages in each of the seven test groups were identified with number tags; the tags bearing numbers from 1 to 4 5 or 46. The first 45 or 46 rats were individually taken out of their delivery boxes and placed in the tagged cages according to the sequence of random numbers. Each animal was then identified by an individual ear tag and cage card. This was repeated for the subsequent three delivery groups, until all 182 rats had been assigned to the seven experimental groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: pharmacologic and toxicologic effects, including behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded at the day of receipt and at days 6, 10, 15 and 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined on days 10, 15 and 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: urogenital tract including uterus and ovaries

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one third of each litter
- Skeletal examinations: Yes: remaining two thirs per litter
- Anogenital distance of all live rodent pups: No

Indices:

No indices calculated. The number of abortions, litters produced, and the mean number per litter or corpora lutea as well as implantations, resorptions, dead and live foetuses and sex ratio were recorded.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals of the highest dose group exhibited some of the following signs: lethargy, scruffy coat, diarrhea, unthrifty appearance, wet bellies, alopecia, soft feces, bloody vaginal discharge, hunched appearance, apparent dehydration.
No such signs were seen in the other dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

There was one treatment-related death at day 18 of gestation and two sacrifices due to apparent signs of abortion (bloody vaginal discharge and weight loss) on day 17 and 18 of gestation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dams receiving the highest dose (512 mg/kg) of Dithane M-45 had a significant, treatment related depression of body weights as compared to the control body weights at 10, 15 and 20 days of gestation. Significantly lower body weights were also recorded at the 128 mg/kg level for days 15 and 20 in comparison to the vehicle control, although they were higher than the body weights of dams at the 512 mg/kg dose. These findings are of toxicological significance and indicate a maternal toxicity at 128 mg/kg. Of the dams carrying fetuses to day 20 of gestation, the only significant difference in body weights minus gravid uteri was a depression at the high dose level (512 mg/kg) as compared to the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a significant, treatment related depression of food consumption in dams receiving the highest dose (512 mg/kg) of Dithane M-45 as compared to control levels of consumption. During gestation days 10-15, dams receiving 128 mg Dithane M-45/kg body weight consumed significiantly less feed than vehicle treated controls but more than dams receiving the highest dose of test material. It is interesting that dams receiving ETU ate significantly more feed during days 10-15 and had a tendancy during the other two periods to eat more food than other groups. This was not accompanied by commensurate increases in body weight. However, the depressed food consumption seen in the high dose test group was associated with a significant depression of adjusted body weights.

Gross pathological findings:

no effects observed

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Two animals of the highest dose group had to be sacrificed due to apparent signs of abortion (bloody vaginal discharge and weight loss) on day 17 and 18 of gestation. The number of abortions and resorptions were significantly increased in the highest dose group.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

The highest dose group was the only dose group were total litter losses by resorption were recorded (9 dams).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The number of resporptions in the highest dose group was higher compared to all other dose groups.
Total number of resorptions/dose group:
83/ 512 mg/kg bw/d;
16/ 128 mg/kg bw/d;
12/ 32 mg/kg bw/d;
14/ 8 mg/kg bw/d;
18/ 2 mg/kg bw/d;
9 / 0 mg/kg bw/d

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pregnat rats of the highest dose group on day 20 had the normal number of live but smaller fetuses.

Dose descriptor:

LOAEL

Effect level:

128 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

32 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: absence of effects

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weights were statistically significantly lower in the highest dose group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Pregnant females of the highest dose group on gestation day 20 had the normal number but smaller fetuses.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Fetuses in the highest dose group exhibited a number of significant skeletal findings including incomplete ossification at several sites, fusion of ribs and vertebrae along with misshapen long bones and vertebral column.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Dams receiving daily doses of 512 mg Dithane M-45/kg body weight produced fetuses which exhibited soft tissue anomalies qualitatively similar to those observed in fetuses taken from dams of the positive control group (50 mg ETU/kg body weight), which includes atrophy of brain tissue, excencephaly and late development of male reproductive organs. In addition, where defects were observed at dose levels of 128 mg/kg or lower, there were either too few incidences to be of any toxicological significance or they occurred across all groups.

Dose descriptor:

LOAEL

Effect level:

512 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

other: increased resorptions

Remarks on result:

other: test material 83% a.i., adjusted to 100% for dosing

Key result

Dose descriptor:

NOAEL

Effect level:

128 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Remarks on result:

other: test material 83% a.i., adjusted to 100% for dosing

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

visceral/soft tissue: central nervous system

Description (incidence and severity):

Mancozeb caused malformations, mainly of the head, at the high dose of 512 mg/kg bw/d.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

512 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the results of this study, a NOAEL of 32 mg/kg bw/d for maternal toxicity and a NOAEL of 128 mg/kg bw/d for developmental toxicity was determined.

Executive summary:

The test substance was administered by gavage in corn oil to groups of 26 primigravid BLU (SD)BR rats day 6-16 of .gestation (day 0, day of insemination) at doses of 0, 2, 8, 32, 128 and 512 mg/kg bw/day. Ethylene thiourea (ETU), a potential metabolite of Dithane M-45, was administered as a positive control at 50 mg/kg bw/day. Rats were observed daily for pharmacologic and toxicologic effects, with weights and food consumption measured on days 6, 10. 15 and 20. All rats were killed on day 20 of gestation and cesarean sections performed. Uterine contents and genital tract were examined for corpora lutea, implantation sites, early and late resorptions, live and dead fetuses. Each fetus was weighed, sexed and examined for gross abnormalities. Approximately one-third of the fetuses were examined for visceral anomalies by the technique of Wilson and two-thirds were examined for skeletal anomalies utilizing Alizarin Red S.

Dithane M-45 at 128 and 512 mg/kg bw/day reduced food consumption and body weight gain of pregnant rats. At 512 mg/kg bw/day the rate of abortion and/or resorptions of litters was significantly increased; however, pregnant rats at day 20 had the normal number of live but smaller fetuses. Also 512 mg/kg bw/day Dithane M-45 increased the frequency of anomalies of the face, head, tail and limbs with exencephaly, hydrocephaly and atrophy of the brain being the major anomalies. ETU at 50 mg/kg b.w./day decreased fetal weights and increased the frequency of anomalies of the face, head, tail, limbs and paws with exencephaly, hydrocephaly and atrophy of the brain being the major anomalies; agenesis and ectopy of the kidney, hydronephrosis, cryptorchisffi and other minor anomalies were also observed.

It is concluded that Dithane M-45 (83% a.i., adjusted to 100% for dosing) is toxic to pregnant rats at 128 mg/kg b.w./day and teratogenic at 512 mg/kg bw/day with 32 and 128 mg/kg bw/day no observed effect levels for maternal toxicity and teratogenicity, respectively. It was further concluded that Dithane M-45 and ETU have similar teratogenic profiles in the.rat, with Dithane M-45 only one tenth as potent as ETU.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

128 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

acceptable for assessment, Guideline and GLP compliant study

Toxicity to reproduction: other studies

Additional information

The following Summary of reproductive toxicity was taken from the  Renewal Assessment Report prepared according to the Commission Regulation (EU) N° 1107/2009 of Mancozeb, Volume1:

 

The reproductive toxicity of mancozeb in mammals was evaluated previously in the original DAR (2000) in studies that were considered to be acceptable. A few publications were identified in the literature but none have produced results that alter the critical NOAEL values presented in the EU Review Report for mancozeb (SANCO/4058/2001-rev. 4.4, July 2009).

Two guideline multi-generational studies (Solomon et al., 1988; Muller, 1992) in the rat were described in the original DAR (2000). Mancozeb had no effect on fertility, reproduction and histopathology of reproductive organs up to doses (approx. 70 mg/kg bw/d) causing generalised and thyroid toxicity (including follicular adenomas mainly in adult males of the F0 and F1 generations) . Pup viability and pup weights were decreased in the second study (Muller, 1992) at approximately 65 mg/kg bw/d, leading to the establishment of a NOAEL for neonatal growth and survival of 7 mg/kg bw/d. This NOAEL was presented and agreed in addendum 3 to the original DAR (2000).

 

Table 1: Overview of regulatory multigenerational studies presented in the original DAR (2000)

Study reference and duration	Species/strain	Test substance/purity (%)	NOAEL (mg/kg bw/d)	LOAEL (mg/kg bw/d)	Effects at the LOAEL
Solomon et al, 1988; 2-gen study	Rat/SD	Mancozeb/84%	70 (reproductive toxicity) 70 (offspring toxicity) 7 (parental toxicity)	> 70 (reproductive toxicity) >70 (offspring development) 70 (parental toxicity)	No effects on reproduction/fertility and offspring development up to top dose Parental toxicity: decreases in body weight and food consumption; effects on organ (liver, thyroid, kidney) weights; thyroid and pituitary histopathology
Muller, 1992 2-gen study	Rat/SD	Mancozeb/88.4%	65 (reproductive toxicity) 7 (offspring toxicity) 7 (parental toxicity)	> 65 (reproductive toxicity) 65 (offspring toxicity) 65 (parental toxicity)	No effects on reproduction/fertility up to top dose; Offspring development (delayed eye opening, decreased weight and viability) Parental toxicity: decreases in body weight and food consumption and thyroid toxicity

 

Five oral developmental toxicity studies (3 in the rat and 2 in the rabbit) were described in the original DAR (2000). These studies were conducted in the 1980s and those performed in the rat used severely maternally toxic maximum dose levels (360 - 512 mg/kg bw/d). These rat studies resulted in malformations (mainly of head and neck) at high doses causing severe maternal toxicity. Mancozeb was classified as Reprotox category 2 (H361d) as a result of these studies. The evidence suggested that the malformations seen in the rat with mancozeb were due to its main metabolite, ETU. ETU is an established developmental toxicant (harmonised classification with Repr Cat 1B2; H3601D) which causes malformations (mainly of head and neck) in the rat in the absence of maternal toxicity. Approximately 7% of mancozeb is converted to ETU in experimental animals (see kinetic section 2.6.1).

New oral regulatory guideline rat developmental toxicity studies on mancozeb (and ETU – Edwards, 2015d) have been conducted by the MTF to provide data on additional endpoints, to clarify the developmental effects attributed to mancozeb and to identify more robust NOAEL values. An oral developmental neurotoxicity study in the rat (OECD TG 426) is also available. This study was conducted to address the concern about a potential relationship between thyroid effects and brain development and was performed in response to a confirmatory data requirement identified following the first review of mancozeb. This study was evaluated by the IT RMS in Addendum 4 (2009) to the original DAR. A few publications were identified in the literature but none have produced results that alter the assessment of this endpoint. The Table below provides an overview of all the available oral developmental toxicity studies. An old published inhalation developmental toxicity study in rats (Lu and Kennedy, 1986), not considered in the original DAR (2000) but described in the recent review of mancozeb by Helath Canada (PRVD2013-01) has also been evaluated. This study is considered to be unreliable due to numerous limuitations.

 

Table 2: Overview of all oral developmental toxicity studies, including literature publications

Study reference, duration and guideline	Species/strain and dose tested (mg/kg bw/d)	Test substance/purity (%)	NOAEL (mg/kg bw/d)	LOAEL (mg/kg bw/d)	Effects at the LOAEL
Gallo et al., 1980; Dev tox study OECD 414	Rat/SD 0, 2, 8, 32, 128, 512	Mancozeb/83%	32 (maternal toxicity) 128 (developmental toxicity)	128 (maternal toxicity) 512 (developmental toxicity)	Maternal toxicity: decreases in body weight and food consumption; Dev toxicity: increased resorptions and malformations mainly of the head;
Tesh et al., 1988; Dev tox study OECD 414	Rat/SD 0, 10, 60, 360	Mancozeb/88.6%	60 (maternal toxicity) 60 (developmental toxicity)	360 (maternal toxicity) 360 (developmental toxicity)	Maternal toxicity: decreases in body weight and food consumption; deaths and paralysis; Dev toxicity: decreased ossification;
Patel, 1999; Dev tox study OECD 414 but of questionable reliability	Rat/Wistar 0, 100, 225, 500	Mancozeb/86.2%	500 (maternal toxicity) 100 (developmental toxicity)	>500 (maternal toxicity) 225 (developmental toxicity)	No maternal toxicity; Dev toxicity: increased malformations (lung emphysema, kidney congestion, dumbbell shaped thoracic centre);
Solomon & Lutz, 1987; Dev tox study OECD 414	Rabbit/NZW 0, 10, 30, 80	Mancozeb/83%	30 (maternal toxicity) 80 (developmental toxicity)	80 (maternal toxicity) >80 (developmental toxicity)	Maternal toxicity: decreases in body weight and food consumption; deaths, abortions and clinical signs No dev toxicity;
Muller, 1991; Dev tox study OECD 414	Rabbit/NZW 0, 5, 30, 55, 100	Mancozeb/88.4%	55 (maternal toxicity) 100 (developmental toxicity)	100 (maternal toxicity) >100 developmental toxicity)	Maternal toxicity: decreases in body weight and food consumption; abortions No dev toxicity;
Beck, 2008b OECD 426 DNT study	Rat/SD 0, 5, 15, 30	Mancozeb/81%	15 (maternal toxicity 30 (DNT))	30 (maternal toxicity) >30 (DNT)	Maternal toxicity: decreases in body weight gain (especially between GD6-12) and thyroid histopathology; No DNT
Edwards, 2015c; Dev tox study OECD 414	Rat/SD 0, 10, 40, 160	Mancozeb/86%	40 (maternal toxicity) 160 (developmental toxicity)	160 (maternal toxicity) >160 (developmental toxicity)	Maternal toxicity: decreases in body weight and food consumption; No dev toxicity;
Axelstad et al., 2011 Perinatal dev tox study (GD7 – PND16) with neurotoxicity investigations Non-guideline	Rat/Wistar 0, 50, 100, 150	Mancozeb/no info on purity	<50 (maternal toxicity) 100 (developmental toxicity)	50 (maternal toxicity) >100 (developmental toxicity)	Maternal toxicity: body weight loss, hind limb paralysis and increased T4 levels No dev neurotoxicity
Hass et al., 2012 Perinatal dev tox study (GD7 – PND16) with sexual development investigations Non-guideline	Rat/Wistar 0, 6.25, 25	Mancozeb/76%	25 (maternal toxicity) 25 (developmental toxicity)	>25 (maternal toxicity) >25 (developmental toxicity)	No maternal toxicity No effects on sexual development
Jacobsen et al., 2012 Peri-/post-natal dev tox study (GD7 – PND16) with thyroid, repro organs and behavioural investigations in offspring Non-guideline	Rat/Wistar 0, 6.25, 25	Mancozeb/no info on purity	25 (maternal toxicity) 25 (developmental toxicity)	>25 (maternal toxicity) >25 (developmental toxicity)	No maternal toxicity No effects on post-natal development
Overgaard et al., 2013 Peri-/post-natal dev tox study (GD7 – PND16) with puberty onset investigations Non-guideline	Rat/Wistar 0, 6.25, 25	Mancozeb/76%	25 (maternal toxicity) 25 (developmental toxicity)	>25 (maternal toxicity) >25 (developmental toxicity)	No maternal toxicity No effects on puberty onset

 

With respect to the evaluation of the developmental toxicity of mancozeb in the rat, it is recognised that the teratogenicity study by Gallo et al (1980) has been central to the decision to classify mancozeb. This study showed that mancozeb caused malformations, mainly of the head, at the high dose of 512 mg/kg bw/d, at which severe maternal toxicity occurred. On the basis of this study, the C&L Specialised Expert Group agreed in 1993 that classification for developmental toxicity was not appropriate because the foetal malformations seen in the rat with mancozeb were attributed to the formation of ETU, a teratogenic metabolite of mancozeb classified with R1B, H360D, but that the levels of ETU produced by mancozeb would not reach the threshold for teratogenic effects. However, the same Group concluded in 2005 that classification of mancozeb with Reprotox category 2 (H361d) was warranted. The Group noted that although the malformations occurred in the presence of severe maternal toxicity (death, paralysis, body weight and food consumption decreases, suffering and total litter loss), they could not be considered the secondary, unspecific consequence of the observed maternal toxicity.

More recent investigations of the developmental toxicity of mancozeb and ETU in the rat, including measurements of plasma levels of ETU (Edwards, 2015c & d) have demonstrated that the foetal malformations observed by Gallo et al (1980) were totally attributable to the production of a teratogenic dose of ETU. The developmental toxicity study of ETU in the rat (Edwards, 2015d) clearly identified 5 mg/kg bw/d as a non-teratogenic dose (NOAEL). Edwards (2015d) also identified 15 mg/kg bw/d as a LOAEL, causing a minimal teratogenic response, whilst 30 mg/kg bw/d was well within the teratogenic dose range. The mean doses of mancozeb calculated to produce blood levels of ETU equivalent to those generated by doses of ETU of 5, 15 and 30 mg/kg bw/d were 143 (range 130-166), 429 (range 390-499) and 859 (range 780-998) mg/kg bw/d respectively. Consequently, when mancozeb was administered to rats at a dose (160 mg/kg bw/d) that was maternally toxic (decreases in body weight and food consumption) but did not exceed the maximum tolerated dose (Edwards, 2015c), insufficient ETU was generated to produce teratogenicity. The UK RMS is of the view that these recent investigations have demonstrated that teratogenic levels of ETU will only be generated at mancozeb doses which cause excessive maternal toxicity. This is because only a small amount (approximately 7%) of mancozeb is converted to ETU in animals; in addition, the rate of this conversion is likely to be slow, such that systemic peaks of ETU are only generated at very high doses of mancozeb. The relevance to hazard identification and classification of such teratogenic effects seen only in the presence of excessive maternal toxicity (death, paralysis, body weight and food consumption decreases, suffering and total litter loss) is questionable. On this basis, classification of mancozeb for developmental toxicity is not warranted and R2 (H361d) should be removed (see CLH dossier for more details).

No developmental neurotoxicity was seen in a regulatory study in the rat up to a dose (30 mg/kg bw/d) which caused maternal toxicity (decreases in body weight gain and thyroid histopathology). A NOAEL for maternal toxicity of 15 mg/kg bw/d was identified from this study. Additional investigations of the developmental toxicity of mancozeb in the rat from the open literature did not identify effects on neurological endpoints, sexual behaviour, post-natal development and puberty onset.

No developmental toxicity was observed in rabbits up to doses (80-100 mg/kg bw/d) causing severe maternal toxicity. Overall, a NOAEL of 160 mg/kg bw/d can be identified for developmental toxicity (Edwards, 2015c) and a NOAEL of 15 mg/kg bw/d can be identified for maternal toxicity (Beck, 2008b). Although there are lower developmental toxicity NOAEL values in other studies, the selected overall NOAEL of 160 mg/kg bw/d is still significantly below the respective LOAEL values. In addition, for some studies the NOAEL is just the highest tested dose at which no developmental toxicity was seen. Furthermore, the selected overall critical NOAEL of 160 mg/kg bw/d is a robust value identified from a modern guideline study. At the expert peer-review meeting 190, it was agreed that the overall NOAEL for developmental toxicity in the rat was 160 mg/kg bw/d (Edwards, 2015a) and 80 mg/kg bw/d in the rabbit (Solomon & Lutz, 1987). The overall NOAEL for maternal toxicity in the rat was 60 mg/kg bw/d (Tesh, 1988) and 55 mg/kg bw/d in the rabbit (Muller, 1991). The experts agreed that the NOAEL of 15 mg/kg bw/d for maternal toxicity in rats from the DNT study (Beck, 2008b) should not be integrated with the NOAEL values from the classical pre-natal developmental toxicity studies due to differences in study design.

No oestrogenic/antiestrogenic activity and no inhibition of aromatase activity were observed in vitro. It is unclear whether mancozeb has antiandriogenic activity in vitro.

 

 

 

Justification for classification or non-classification

The following conclusion was taken from the RAC Opinion proposing harmonised classification and labelling at EU level of Mancozeb (March 2019):

Conclusion on classification

According to the CLP criteria, classification in Category 1A is based on evidence from human data. No evidence of association between reproductive toxicity and exposure to mancozeb in humans is available. Therefore, classification as Repr. 1A is not warranted.

Categories 1B and 2 are reserved for presumed and suspected human reproductive toxicants respectively, and classification in these categories is based on the presence of ‘clear’ (Category 1B) or ‘some’ (Category 2) evidence of an adverse effect on sexual function, fertility, or development. In addition, such evidence must be present in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effects on reproduction must be considered not to be a secondary non-specific consequence of the other concurrent toxic effects.

RAC concludes that mancozeb meets the criteria for classification in Category 1B due to clear developmental findings in rats considered not to be a secondary non-specific consequence of the other concurrent toxic effects. The following considerations have been summarised in the opinion:

 

• Mancozeb induced severe malformations in the Anon. (1980) study at a maternally toxic dose of 512 mg/kg bw/d. According to CLP criteria (3.7.2.3.5), the presence of maternal toxicity shall not be used to negate findings of embryo/foetal effects, unless it can be clearly demonstrated that the effects are secondary non-specific effects. This is especially the case when the effects in the offspring are significant, e.g. irreversible effects such as structural malformations. As summarised above, RAC considers that the developmental effects observed in Anon. (1980) are severe and irreversible. The WoE indicates that they occurred due to a direct action of mancozeb and/or its major metabolites (including ETU) on the foetuses and are not related to the excessive maternal toxicity observed at 512 mg/kg bw/d.


• In relation to the Anon. (1980) study, RAC has the following additional concerns:

o Single occurrences of severe and rare external and visceral malformations also occurred at 128 mg/kg bw/d in the same study indicating that this dose, associated with only limited maternal toxicity, lies close to the threshold dose causing malformations;

o ETU was used in the study as a positive control group to compare with mancozeb since embryo/foetal effects were observed in earlier independent studies with ETU in rats (e.g. Khera, 1973; Teramoto et al., 1978; Chernoff et al., 1979). The spectrum of malformations with mancozeb was similar to that in the ETU-treated group, where no maternal toxicity was observed.

• A single dose of 30 mg/kg bw/d ETU on GD 15 induced severe dilation of brain ventricles due to necrosis of brain tissue (Khera and Tryphonas, 1977). Although the study has been performed before GLP, RAC notes that the study has been well-conducted and it does not reduce the concern about developmental effects of mancozeb.


• The mode of action (MoA) of mancozeb and/or its major metabolites (including ETU) behind the hydrocephalus in rats is not fully established. The pattern of findings of embryo/foetal effects is complex. Mechanistic studies indicate that cell necrosis in the central nervous system is part of the MoA (Teramoto 1978b; Khera and Tryphonas 1977; Khera 1987).


• RAC notes the equivocal PNDT study of Anon. (1988c) with reduced ossification of the skull and of the thoracic vertebra at 360 mg/kg bw/day. The increased incidence of incomplete ossification of the interparietal bone might be related to meningoencephalocele observed at a higher dose in the study by Anon. (1980) (cf. Khera, 1973).


• RAC is of the opinion that humans resemble the rat species in their ability to metabolise mancozeb to ETU.

RAC also notes additional negative PNDT studies in rats (Anon., 1999b; Lu and Kennedy, 1986) and in rabbits (Anon., 1987b; Anon., 1991b). RAC questions the validity of the negative rat PNDT study of Anon. (1999b), where no maternal toxicity was observed at doses up to 500 mg/kg bw/d.

In the most recent PNDT study of Anon. (2015d), no developmental effects were seen. However, maternal toxicity at the top dose of 160 mg/kg bw/d was rather limited and the preliminary study in non-pregnant animals (Anon., 2015b) indicated that a higher dose could have been tested. Therefore, due to this selection of the top dose, the Anon. (2015d) study does not adequately address the developmental toxicity concerns raised by the previous Anon. (1980) study.

RAC acknowledges the comments provided during the public consultation (comment No 17 in the RCOM table) referring to the doses used in the studies Anon. (2015c & d) that were probably chosen too low to cause effects on the foetal development. The comments further noted that with regard to the dose range finding study (Anon., 2015b), doses up to 240-300 mg/kg bw/d mancozeb could be possible without causing severe maternal toxicity in the animals.

In conclusion, RAC considers that the new data is not convincing enough to reduce the concern for the malformations seen in the original Anon. (1980) study. Therefore, removal of the current classification in Category 2 proposed by the DS is not considered appropriate. Moreover, the severe and irreversible developmental findings in Anon. (1980) make it difficult to argue for a category 2 classification. There is no mechanistic data available to indicate specific maternally-mediated mechanisms that give rise to secondary developmental effects in the offspring. The lack of connection between the maternal toxicity and severe malformations in the rat study Anon. (1980) leads RAC to conclude that mancozeb meets the criteria for classification in Category 1B for adverse effects on development.