Desmedipham

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-01-06 to 2015-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2000 draft

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Desmedipham

EC Number:

237-198-5

EC Name:

Desmedipham

Cas Number:

13684-56-5

Molecular formula:

C16 H16 N2 O4

IUPAC Name:

ethyl 3´-phenylcarbamoyloxycarbanilate

Test material form:

solid: particulate/powder

Details on test material:

The test material is Desmedipham.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: Males: 155-162 g; females: 139-149 g
- Housing: Solid floor Polypropylene cages (410 x 282 x 180 mm).
- Diet: Ground diet
- Water: Unlimited supply of water filtered through a Aquaguard water filter system.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 64 %

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

desmedipham was dissolved in acetone before being added to the powder diet, in control diet only acetone was added

Details on exposure:

PREPARATION OF DOSES:

DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diet was prepared once in three or four days throughout the experiment period based on the stability data.

Experimental Diet Preparation
Experimental diets for each group of animals was prepared in quantities of 4 - 12 kg, per mixing. The requisite quantity of desmedipham and dissolved in acetone. As the test substance was not easily miscible in water, acetone was used to facilitate the test substance mixing in experimental diet. The respective desmedipham/acetone solutions were then added to the powder diet (in case of control group only acetone was added) to produce the requisite test diet. Mixing was performed with a double cone blender or a "Y"- shaped blender for 20 minutes and the resulting test diet duly transferred to polythene bags after allowing acetone to evaporate within labeled stainless steel containers and stored in the experimental room. The experimental diet was tested regularly, once in two months intervals for homogeneity of mixing.

VEHICLE
The test substance will be dissolved in a minimum quantity of Acetone (vehicle) before mixing with the diet. The control animals will be given diet mixed with an equivalent amount of vehicle (Acetone) only.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After two\three weeks mating period or 3 estrous period of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually in solid floor polypropylene cages.
- For all P and F1 males sperm analysis were performed at termination. Sperm were analysed for epididymal sperm morphology, homogenization resistant testicular sperm head count, epididymal sperm count and sperm motility.

Analytical verification of doses or concentrations:

yes

Remarks:

According to analysis actual concentration of desmedipham in the diet varied within 92.7% of nominal concentration.

Details on analytical verification of doses or concentrations:

The test substance present m known quantity of the test diet (10 g) was soaked in 50 ml of ethyl acetate and filtered and concentrated to dryness in a rotary vacuum evaporator at 45°C. The residue obtained was dissolved in 10 ml of HPLC acetonitrile and analysed in HPLC employing SPD1 OA UV detector.

Duration of treatment / exposure:

For parental animals: at least 70 days prior to mating and continued up to the day of sacrifice.
Treatment of the F1 animals selected for siring the next generation (i.e. F2 generation) will begin after weaning and end at weaning of F2 pups.

Frequency of treatment:

Continuously

Details on study schedule:

The test substance (desmedipham technical) was provided m experimental diet for 70 consecutive days continuously (7 days a week) to four dose groups (0, 50,250 and 1 250 ppm) of rats (each group comprising of 24 males and 24 females rats) prior to their mating and then through mating, parturition and weaning of the F1 generation pups. The rats were mated in a 1:1 ratio to produce the F1 generation. Each morning the females were examined for the presence of sperm and the day designated as day 0 of pregnancy (the day on which sperms are observed in the vaginal smears of rat). Sterilized paper cuttings were provided on day 14 of gestation as nesting material to the dams. All pups were observed daily for physical developmental landmarks. The pups were subjected to sensory reactivity to stimuli, motor activity on lactation day 20. One male and one female pup were randomly selected from each dam, wherever possible. Females failing to mate were provided with additional opportunities to mate with other proven sires, failing which animals were euthanised by an overdose of carbon dioxide for histopathological examination of the reproductive organs. To obtain rats for the next generation, rats from the F1 generation were randomly selected (wherever possible one male and one female per dam) and subsequently mated in a 1 : 1 ratio to produce the F2 generation, which is terminated on day 21 after littering. All the parents (P) and F1 pups not selected for siring the next generation were sacrificed by an overdose ofC02 during, the post partum period. Dosing of the F1 animals selected for siring the F2 generation continued during their growth into adulthood and then through their mating (sibling mating was avoided), parturition and weaning of the F2 generation pups. All the F1 animals and F2 generation pups were sacrificed on day 21 of the post partum period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

50 ppm

Doses / concentrations 3

Dose / conc.:

250 ppm

Doses / concentrations 4

Dose / conc.:

1 250 ppm

No. of animals per sex per dose:

24/sex/group

Control animals:

yes

Positive control:

Not required

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment and weekly thereafter. Body weight of pregnant females was recorded on Days 0, 7, 14 and 20; and on Days 0, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, During the pre mating and gestation periods, cage-wise feed consumption was measured weekly at a minimum. After parturition and during lactation, feed consumption measurements was made on the same day as weighing of the litters. Weekly feed consumption of the parental male animals was not recorded after mating.

Feed spillage/wastage was not estimated during the course of the study and evidence of excessive spillage was documented in the raw data.

- Feed efficiency (body weight gain per gram of feed consumed) and test substance intake for P and Fi animals, was calculated based on feed consumption and body weight data and appended in the report accordingly.

Oestrous cyclicity (parental animals):

Estrous cycle length and pattern was evaluated by vaginal smears for all P and F1 females for 22 days prior to cohabitation. Care was taken to avoid disturbance of mucus while obtaining vaginal smear. Ovarian cyclicity was monitored by observing changes in the vaginal cytology. Daily morning vaginal smear was collected and observed using microscope from each female rat for 22 days prior to cohabitation. Based on the stage the cycling pattern was determined. Following the attainment of puberty, estrous (heat) occurs in the non pregnant female in a species -specific rhythmic cycle. The time period between the onset of one estrous and the next is defined as the estrous cycle. It is characterised by four major phases: Proestrous, estrous, metaestrous and diestrous. During cohabitation period vaginal smear were performed to confirm the mating. Prior to termination of female rats vaginal smear was performed to check the stage.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testis weight, epididymis weight, sperm motility, epididymal sperm count, sperm morphology and homogenisation resistant testicular spermatid head count.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, on lactation day 4
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Partial adjustment (eg. Five males and three females) was performed. Adjustment was not done for litters of eight pups or less.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
Physical Developmental Landmarks, Sensory Reactivity of Stimuli, Click Response, Tail-pinch Response, Pupil Response, Air Righting Reflex, Motor Activity, Pup Body Weight

GROSS EXAMINATION OF DEAD PUPS:
Yes, Dead pups when not macerated or pups that are killed in a moribund condition was examined and preserved.

Postmortem examinations (parental animals):

SACRIFICE
All P and F1 adult males and females were sacrificed at termination.

GROSS NECROPSY
Gross pathological observations was made for all parental animals (P and F1). Special attention was paid to the organs of the reproductive systems. At the time of necropsy, vaginal smear was examined to determine the stage of estrous cycle. The uterus of all cohabited females was examined for the presence and number of implantation sites.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues or representative samples were fixed and stored in a suitable medium for examination.
Vagina, uterus with oviducts, cervis and ovaries.
One testis, one epididymis, seminal vesicle, prostate and coagulating gland.
Pituitary and adrenal gland.
Vital organs, from all P and F1 animals selected for mating.
Gross lesions.
Full histopathology of the mentioned organs was performed for all high and control P and F1 animals selected for mating. Organs with treatment related changes were also examined in the low and mid dose groups.
Organ Weights: The weight of the following organs was recorded for all prenatal and F1 generation animals selected for siring the F2 generation: adrenals, brain, ovaries/testes, kidneys, liver, spleen, uterus (with oviducts and cervix) epididymides (total and cauda), seminal vesicle with coagulating and their fluid (as one unit), prostate, thyroid and pituitary. Paired organs were weighed individually.

Postmortem examinations (offspring):

SACRIFICE
F1 offspring not selected for mating and all F2 offspring were sacrificed at weaning.
These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Both the F1 and F2 weanlings were examined macroscopically for any structural abnormalities or pathological changes. Special attention was paid to the organs of the reproductive systems. Dead pups when not macerated or pups that are killed in a moribund condition was examined and preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
For F1 and F2 weanlings, gross lesions of one pup/sex per litter from both F1 and F2 generation.
Organ Weights: For F1 and F2 weanlings, the following organs were weighed for one randomly selected pup per sex per litter: brain, spleen and thymus.

Statistics:

Statistical analysis was carried out for the parameters using validated software. Non-pregnant females animals were not considered for statistical analysis. The parametric data (body weight, body weight gain, feed consumption, feed efficiency, sperm parameter, estrous cycle parameter, physical and sexual developmental landmark, litter size, organ weight, and relative organ weight) were analysed by using Bartlett's test of homogeneity of variance. Where the result was not significant then analysis of variance (ANOVA) was carried out. If ANOVA was significant then Dunnett's multiple comparison tests were carried out. If Bartlett's test of homogeneity was significant then a student's "t" test was carried out. The non-parametric data (mortality rate, pregnancy rate, male:female ratio, male fertility index, female fertility index, live birth index, survival index, gestation index, and lactation index) were analysed using Chi-square test.
See "Any other information on materials and methods incl. tables" for more information.

Reproductive indices:

Male and female fertility index; gestation index; lactation index and parturition index were calculated.

Offspring viability indices:

Live birth index and survival Indices were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed during the study.
Male: All the experimental rats were observed once daily for onset of clinical signs, majority of the animals appeared normal during the experimental period. The signs such as soft stool (during Days 35-37 and 42-44) and diarrhoea (Days 42-44, 47-50 and 58-61) transient in nature were observed in few animals for a few days in all experimental groups. Emaciation was observed in three animals (Days 42-44) and wry neck (Days 35-37 and 91-92) for one animal in control group only. Lacrimation, nasal discharge, and emaciation was observed (Day 63-112 in one animal at 50 ppm only. From the above observation it is evident that no treatment related, clinical symptoms were exhibited by desmedipham technical up to the dose level of 1250 ppm.
Female: Clinical symptoms such as soft stool, lethargy and emaciation transient in nature were recorded in few females at 50 ppm dose group only. Hard mass on right side of abdomen was observed in one control animal on Days 101-115. As no other clinical symptoms were observed in 250 and 1250 ppm dose group, it is concluded as these were not treatment related effect.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No treatment-related mortalities were observed during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: No significant effect on body weight were recorded during the pre-mating period. However, significant marginal reduction in body weight of treated groups were observed at 250 ppm during Weeks 12-16 and at 1250 ppm on Weeks 11-12 only. During the remaining experimental period, body weights were comparable with controls and the corresponding body weight change were not observed during the same period. So it is considered that the male animals were not affected by treatment with desmedipham technical up to a dose level of 1250 ppm.
Females: The weekly body weight of females, recorded during the ten weeks of premating period were comparable between the control and treatment groups up to the dose level of 250 ppm. Treatment related significant reduction in body weight were observed from Week 5 onwards at 1250 ppm.
Gestation Body Weight:
The body weight of pregnant animals was recorded on Days 0, 7, 14 and 20 of gestation. A significant reduction (marginal) was observed at 50 ppm level at gestation Days 14 and 20, which is considered as incidental as no effect was observed at 250 ppm. Treatment-related significant reductions in body weight were observed throughout the gestation period at 1250 ppm.
Lactation Body Weight:
The body weight of female rats was recorded on lactation Days 0, 4, 7, 14 and 21. Data for rats at 50 and 250 ppm are comparable to controls. Significant reductions in body weight were observed on lactation Days 0, 4, 7 and 14 at 1250 ppm, compared to controls.
Percent body weight change:
Males: Significant reductions in per cent body weight change were observed in all treated groups at Week 10, at 50 ppm at Week 4 and at 1250 ppm at Week 14. Significant increases observed at 50 and 1250 ppm at Week 12 are considered to be incidental.
Females: The weekly per cent body weight change of female rats were calculated during the ten weeks of premating period. Significant reductions in percent body weight change were observed in all treated groups at Week 8; at Week 5 at 50 and 1250 ppm, and at Weeks 2 and 3 and are considered to be incidental.
Gestation: The percent body weight of pregnant animals was calculated during gestation Days 0-7, 7-14 and 14-20. No effects of treatment were noted.
Lactation: The body weight change of female rats was recorded on Days 0-4, 4-7, 7-4 and 14-21 of lactation. Significant increase in percent body weight change were observed at 250 and 1250 ppm during lactation Days 0-4 when compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect on the food consumption of the parental generation animals during the whole experimental period.
Pre-mating Period:
Male: The feed consumption (including spillage) pattern of male rats remain unaffected up to a dietary concentration of 1250 ppm during the ten weeks of premating period except a significant increase observed at 250 ppm on 6th week, which is considered as incidental and not treatment related.
Female: The feed consumption of female rats did not appear to be affected, up to a dietary concentration of 1250 ppm during the ten weeks of premating period except for a significant reduction observed at 50 ppm during Week 9 which is considered as incidental and not treatment related.
Gestation Period:
The feed consumption recorded during the period of gestation was comparable between the control and treated groups except for a significant increase observed during gestation Days 7-14 only at 1250 ppm, which is considered as incidental.
Lactation Period:
The feed consumption recorded during the period of lactation was comparable between the control and the treated groups except for a significant decrease observed on lactation Days 7-14 at 50 ppm, considered as not treatment related.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

For the P generation decreases in feed efficiency were observed in males at 1250 ppm and 250 ppm but only during the first weeks (15-18%). In P generation females, decreased feed efficiency was observed in the high dose (30-51%) during weeks 1-10 and some increases and decreases were also seen during lactation and gestation, which seemed spurious.
Pre-mating Period:
Male: The feed efficiency of male experimental animals did not appear to be affected, up to a dietary concentration of 50 ppm during the premating period. Significant decrease in feed efficiency was observed on experimental Week 1 and 2 for the 250 and 1250 ppm dose groups. During the remaining experimental period (from week 3 onwards) the values were comparable to that of the control group.
Females: The feed efficiency of female experimental animals was significantly lower in Weeks 2, 3, 5 and 8 at 1250 ppm. Significant decrease in feed efficiency was observed in Week 8 in 50 and 250 ppm. Values for the remaining period were observed to be comparable to the control group.
Gestation Period:
The feed efficiency recorded during the period of gestation was comparable between the control and treated groups except a significant increase observed during gestation day 14-20 at 250 ppm and decrease during 7-14 of gestation day at 1250 ppm.
Lactation Period:
The feed efficiency recorded during the period of lactation was comparable between the control and the treated groups except a significant increase observed during lactation day 0 - 4 at 250 and 1250 ppm and also during lactation day 14-21 in 50 and 1250 ppm dose groups. There was no consistent treatment related effect observed.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination in different organs belonging to different experimental groups revealed varying degree of lesions in lungs (congestion / haemorrhagic spot, pneumonic changes / mycoplasmosis, medial hypertrophy of muscular arteries); spleen (lymphoid hyperplasia, deposition of haemosiderin pigment, extramedullary haematopoietic foci megakaryocytosis); kidneys (inflammatory changes, congestion, cystic dilation of tubules); liver (degenerative /necrotic changes, extramedullary haematopoietic foci); heart (chondroid metaplastic focus in aortic wall, segmental degeneration / necrosis of myofibrils); brain (ependymal cell hyperplasia, ventricular dilation, congestion); pituitary (Rathke's cleft with / without secretion, hyperplasia / hypertrophy, bony metaplastic focus in pars distalis); thymus (congestion/ haemorrhagic spot, atrophy depletion of lymphoid elements, sinus histiocytosis); adrenals (congestion, cortical vaccuolation, accessory cortical nodule); thyroid (hypertrophy / hyperplasia of follicular lining cells, ultimobranchial cyst); epididymis (vacuolation of lining epithelium) and uterus (pigmentation in myometrium, hydrometra / luminal dilation).

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Three weeks prior to cohabitation, a vaginal smear was prepared daily for all female rats to detect the stages of oestrous cycle. It was observed that all female rats were exhibited oestrous prior to the day of cohabitation. Desmedipham does not produce any treatment related adverse effect on oestrous cycle up to the dose level of 1250 ppm.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm count: Statistically significant reductions in the percent motile sperm, number of epididymal sperm count and testicular sperm head count were observed at 1250 ppm. The testicular sperm head count was significantly lower at 50 and 250 ppm when compared to controls. The observed reductions were minimal in number and there was no effect on fertility up to the dose level of 1250 ppm.
Sperm Morphology: The sperm morphological changes such as detached head and folded tail were significantly higher; short and blunt head, coiled tail, folded tail, and broken tail were observed significantly lower in 50 and 250 ppm and were not observed at 1250 ppm dose level. Other observed changes such as blunt head, reduced hook, head less tail and bent tail were considered as biologically not significant and not related to treatment up to 1250 ppm.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility:
Male and female fertility index, gestation and lactation index were comparable between the control and the treated groups. Pregnancy rate and parturition index were not affected by treatment. The duration of the gestation was comparable among all the dose groups i.e., treatment with desmedipham technical up to a dietary concentration of 1250 ppm did not influence fertility.
Live Birth Index and Survival Indices:
Mean number of live pups, sex ratio, pups’ survival index and live birth index were comparable among all the treated groups. No treatment related effects were observed up to the dose level of 1250 ppm.
Number of Pups:
Numbers of pups were recorded on lactation days 0, 4, 7, 14 and 21. Total (Male + Female), female pup number were comparable between the control and treated groups. Mean litter size of male pups was significantly lower at 50 ppm on lactation days 7,14 and 21. Such a reduction was not observed at 250 and 1250 ppm, so it is not considered as a treatment-related effect.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Equivalent to 4 mg/kg bw/day

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Males: The majority of the animals appeared normal during the experimental period. The symptoms of soft stool transient in nature were observed in few animals for few days in all experimental groups. Bronchial rale was observed during Day 159-161 for one animal in control and one from 1250 ppm dose group only. Soft stool and nasal discharge, emaciation and bronchial rale, soft stool and bronchial rale was observed in one animal at 250 ppm only. From the above observations it is evident that no treatment related, clinical symptoms were exhibited by desmedipham technical up to the dose level of 1250 ppm.

Females: Clinical signs of emaciation transient in nature were recorded in one female at 50 ppm only during Days 194-197. Wry neck was observed in one animal during Days 196-241 at 250 ppm. As no other clinical symptoms were observed up to 1250 ppm, it is concluded that these were not treatment-related effects.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two male mortalities were observed at 250 ppm. Two female mortalities, one each from controls and 1250 ppm were observed during the experimental period. These mortalities were not considered to be a treatment-related effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of male rats remained unaffected up to the dietary concentration of 1250 ppm.
Treatment-related reductions in the body weight of females was observed at 1250 ppm during the premating, gestation and lactation periods.
Body weight:
Males: The weekly body weight of males during the eleven weeks of the pre-mating period were comparable between the control and treatment groups up to the dose level of 1250 ppm.
Females: The weekly body weight of female experimental animals, recorded during the eleven weeks of premating period were comparable between the control and treatment groups up to 50 ppm. Treatment related significant reduction in body weight were observed from Week 3 at 1250 ppm and Week 9 at 250 ppm.
Gestation Body Weight:
The body weight of pregnant animals was recorded on gestation Days 0, 7, 14 and 20. Significant reductions were observed at 250 ppm on gestation Days 0 and 7. Treatment related significant reductions in body weight were observed throughout the gestation period at 1250 ppm.
Lactation Body Weight:
The body weight of female rats was recorded on lactation Days 0, 4, 7, 14 and 21. The data from control and 50 ppm treatment groups were comparable. Significant reductions in body weight were observed on lactation Days 0, 4 and 7 at 250 ppm, and throughout the lactation period at 1250 ppm.
Percent body weight:
Males: Significant marginal reductions in percent body weight were observed at 250 and 1250 ppm during Week 2. Significant increases in percent body weight were observed in Week 8 at 50 ppm and Weeks 3 and 7 at 1250 ppm. During the remaining study period, percent body weight changes were comparable with control group. It is concluded that males were not affected by treatment with desmedipham technical up to 1250 ppm.
Females: Significant reductions in percent body weight were observed at 250 ppm during Weeks 1-4 and at 1250 ppm during Weeks 1, 2 and 10. These changes were not consistent and corresponded to body weight reduction observed in treated groups.
Gestation:
Treatment related significant effects on body weight change were not observed throughout the gestation period up to the dose level of 1250 ppm.
Lactation:
Body weight changes for female rats were recorded on Days 0-4, 4-7, 7-14 and 14-21 of lactation days. The data from all groups were comparable.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption remained unaffected up to a dietary concentration of 1250 ppm. No major impact on feed efficiency was observed during the experimental period.

Pre-mating Period:
Males: Food consumption (including spillage) pattern of male experimental animals remained unaffected up to a dietary concentration of 1250 ppm during the premating period.
Females: Food consumption of female experimental animals did not appear to be affected, up to a dietary concentration of 1250 ppm during the eleven weeks of premating period except for a significant reduction in Week 9 at 250 ppm and increase in Week 4 at 1250 ppm which are considered incidental and not treatment related.
Gestation Period:
Food consumption recorded during gestation period was comparable between the control and treated groups.
Lactation Period:
Food consumption recorded during lactation was comparable between the control and the treated groups except for a significant decrease on lactation Days 4 -7 at 250 ppm and significant increase on lactation Days 7-14 and 14-21 at 50 ppm, considered as incidental in nature.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Pre-mating period:
Males: Food efficiency in males appear to be affected, up to a dietary concentration of 1250 ppm during the premating period. Significant decreases in feed efficiency were observed only in Week 6 at 1250 ppm and significant increases in Week 4 at 50 ppm Week 7 at 1250 ppm.
Females: Food efficiency in females was not significantly affected. A significant decrease in feed efficiency was observed in Week 10 at 1250 ppm; values for the remaining period were observed to be comparable to controls.
Gestation Period:
Food efficiency recorded during gestation was comparable between the control and treated groups except for a significant decrease on gestation Days 0-7 at 50 and 1250 ppm.
Lactation Period:
Food efficiency recorded during lactation was comparable between the control and the treated groups.

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:

effects observed, treatment-related

Description (incidence and severity):

Terminal Body Weight and Organ Weight:
Males: Terminal body weights of treated male animals was observed to be comparable to that of control group. Absolute kidney weight was significantly reduced at 1250 ppm only. Significantly higher spleen weight was observed at 1250 ppm only. Findings are considered to be treatment related. No changes were observed in the weights of the adrenals, testes, epididymis, cauda epididymis, prostate, seminal vesicle and coagulating gland, thyroid and parathyroid, brain, pituitary, heart, liver, and thymus.
Females: A significant reduction in terminal body weight of female rats was observed only at 1250 ppm. No significant variation was observed in the weights of the ovaries, uterus, brain, pituitary, heart, thymus, and liver. A significant increase was noticed in the weights of the thyroid and parathyroid at 1250 ppm only. A significant reduction in the weight of the adrenals at 1250 ppm, kidney at 250 and 1250 ppm and spleen at 50 ppm were also observed.
Organ Weight Body Weight Ratio:
Males: Terminal organ weight body weight ratio of male animals was found not affected by the treatment at 50 and 250 ppm dose groups. Data on the relative organ weight revealed that the relative organ weight of the kidney was significantly lower, spleen and brain were significantly higher at 1250 ppm dose level only when compared to control group. No other significant changes were observed.
Female: No significant changes in ratio was observed up to the dose level of 250 ppm except, significant decrease in ratio of spleen at 50 ppm dose group. Significant increase was noticed in the organ weight ratio of thyroid and parathyroid and brain at 1250 ppm.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathological observation did not reveal any treatment related changes in the animal at any dose level.
The external examination of the carcasses did not show any significant lesion / abnormality. Like parental generation certain lesions were seen in lungs (congestion / haemorrhagic spot, emphysema, pneumonic foci, hepatization, abscess, oedema); kidneys (congestion, pallor, mottling); spleen (mottling, white granular deposits, enlargement atrophy, blackish discolouration); liver (pallor, mottling, congestion, nodular growth, parasitic cyst, hepatomegaly); thymus (enlargement, congestion / haemorrhagic spot); testis (abscess); epididymis (abscess); uterus (hydrometra, pyometra); ovary (cystic ovarian bursa) in some of the animals without any sex / group biasness.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologically, varying degree of congestion, deposition of hemosiderin pigments and extramedullary hematopoiesis, mainly at the highest dose, were observed in the spleen of treated animals belonging to first filial generation rats. Important observations comprised of lesions in lungs (congestion / haemorrhagic spot, pulmonary mycoplasmosis / pneumonia, medial hypertrophy of muscular arteries); spleen (lymphoid hyperplasia, congestion, haemosiderin pigments, metaplastic focus); heart (chondroid-^.'metaplastic focus); kidneys (mottling / congestion); brain (submeningial congestion, hyperplasia of choroid plexus); pituitary (congestion, hyperplasia of acidophilic cells); thymus (congestion / haemorrhagic spot, lymphoid depletion); adrenals (congestion, hypertrophic foci in cortex, accessory cortical nodule); thyroid (follicles devoid of colloids, ultimobranchial cyst, follicular lining cell hyperplasia); prostate (hyperplasia of acinar lining epithelium / papillary projection); uterus (luminal dilation, deposition of pigments in myometrium) and mammary gland (ill-developed, hypertrophy / hyperplasia of acinar lining cells).

The value of different follicles (primordial, growing, primordial + growing and antral) in control and high dose (1250 ppm) groups did not differ significantly.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Three weeks prior to cohabitation vaginal smears were prepared daily in all female rats to detect the stages of the oestrous cycle. It was observed that all female rats were exhibited the oestrous stage prior to the day of cohabitation. The test substance does not produce any treatment related adverse effect on oestrous cycle up to the dose level of 1250 ppm.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm Number:
Statistically significant reductions in percent motile sperm and number of epididymal sperm count were observed at 1250 ppm. The epididymal sperm count were significantly lower at 250 ppm when compared to controls. Percent motile sperm was significantly lower at 50 ppm. There were no significant changes observed in number of testicular sperm head count in treated groups. The reductions observed were minimal in number and an effect on fertility was not affected up to the dose level of 1250 ppm.
Sperm Morphology:
Changes such as short head, broken tail were significantly higher at 50, 250 and 1250 ppm. Other observed changes such as tail-less head, pin head, coiled tail and wavy tail were considered as biologically not significant and not related to treatment effect up to the dose level of 1250 ppm.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility:
Male and female fertility index, gestation index and lactation index were comparable between the control and the treated groups. Pregnancy rate and parturition index were not affected by the treatment. The duration of gestation was comparable among all the dose groups i.e., treatment with desmedipham technical up to the nominal concentration of 1250 ppm did not influence fertility.
Live Birth Index and Survival Index:
Mean numbers of live pups, sex ratio, pups’ survival index and live birth index were comparable among all groups. No treatment related effects were observed up to 1250 ppm.
Number of Pups:
The total number of (Male +Female) pups, number of male pups and female pups were comparable between the control and treated groups.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Equivalent to 4 mg/kg bw/day

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In female pups, the number of days for hair growth was significantly lower at 1250 ppm dose group when compared to control group. The changes were observed in number of days were minimal in numbers and the values were within normal biological variation. So these changes were not considered as treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant change was observed in pup mortality during the lactation period in male, female and total (male + female) pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Pup weights were recorded on lactation Days 0, 4, 7, 14 and 21. Total litter weight of (male and female) pups at 50 ppm dose group was reduced on Day 4 and increased on Day 21 when compared to controls. Significant reductions in mean litter weight were observed on lactation Days 0 and 4 at 250 ppm dose group, and on lactation Day 14 only at 1250 ppm.
Sex-wise analysis indicated that the litter weight (mean) of male pups at 50 ppm was significantly reduced on Day 4 and increased on Day 21. Mean pup weights on Days 0 and 4 were reduced at 250 ppm for male pups.
Significant increases in female pup weight were observed on lactation Day 21 at 50 ppm. At 250 ppm, a significant decrease in body weight was observed on Day 0 and in increase observed on Day 21 when compared to control group pups. However, these changes were not noticed at 1250 ppm; hence it cannot be considered as a treatment-related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on food consumption by F1 offspring.

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

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

In male pups, the number of days for balanopreputial separation was significantly higher at 1250 ppm when compared to the control group. The changes observed in number of days were minimal in number and the values were within the normal biological biological variation. The mean body weight at balanopreputial separation day of treatment groups was comparable with current control group.
In female pups, the number of days for vaginal opening of treated groups was comparable with concurrent control group. The mean body weight at vaginal opening day of treatment groups was comparable with the concurrent control group.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Terminal body weight and organ weight:
Males: Terminal body weight of male pups and absolute weight of brain, spleen and thymus of treated dose groups were comparable to that of the control group.
Female: No significant changes were observed in terminal body weight and the organ weight of female pups up to the dose level of 1250 ppm.
Organ Weight-Body Weight Ratio of Pups:
Male Pup: Relative weight of brain, spleen and thymus of pups in all treated groups were comparable to that of the control group.
Female pup: No significant variation was observed in the ratio of various organs up to the dose level of 1250 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross pathological observation did not reveal any treatment related changes in the animal and pups at any dose level.

Histopathological findings:

no effects observed

Description (incidence and severity):

No effects of treatment were observed.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Physical Developmental Landmarks:
Male pups: Significant increase-in number of days for ear opening was observed at 50 ppm. Number of days for unfolding of pinna, tooth eruption and eye opening were significantly higher at 250 ppm. Significant increase in number of days for eye opening and decrease in number of days for hair growth were observed at 1250 ppm when compared to control group. The above changes were observed to be of minimal variation in number of days and within the normal biological variation.
Female pups:
Observations at 50 ppm were comparable to the control group. The number of days for unfolding of pinna, tooth eruption, eye opening and hair growth were significantly higher at 250 ppm. The number of days for eye opening and vaginal opening were significantly higher and significantly lower for hair growth at 1250 ppm when compared to control group. The changes observed in number of days were minimal in number and the values were within the normal biological variation.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Sensory Reactivity to Stimuli:
Males: All pups from control and treated dose groups showed presence of auditory startle, normal pupil response and normal air righting reflex. The majority of the pups exhibited tail pinch response as "flinch" and few pups from all dose groups showed "slight" reaction for tail pinch response. There was no treatment related effect observed up to the dose level of 1250 ppm.
Females: All the pups from control and treated dose groups showed presence of auditory startle, normal pupil response and normal air righting reflex. Majority of the pups from treatment and control groups revealed "flinch" to tail pinch response during the experimental period, few pups showed "slight" response. There were no treatment related changes observed up to the dose level of 1250 ppm.
Motor Activity:
Males: Significant increase in the total motor activity, ambulatory activity and stereotypic activity were observed in all treated groups (male pups) during 0-5 minutes and 5-10 minutes only at 1250 ppm dose group when compared with control group. During 10-15 minutes observation the values of treated groups were compared to control group.
Females: Treatment of desmedipham technical up to the dose level of 1250 ppm did not cause any significant alterations in total motor activity, ambulatory and stereotypic activity of female pups as compared to the control group.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Eye opening was slightly but significantly delayed in male and female F1 offspring at 250 and 1250 ppm in the absence of effects on bodyweight. Sexual maturity was delayed at 1250 ppm.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Eye opening was slightly but significantly delayed in male and female F1 offspring at 250 and 1250 ppm

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In female pups, the number of days for hair growth was significantly lower at 1250 ppm compared to controls. The changes were observed in number of days were minimal in numbers and the values were within normal biological variation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant change was observed in pup mortality during the lactation period in male, female and total (male + female) pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight of male and female pups at 50 ppm was found to be not affected during lactation when compared to controls. Significant reductions in mean litter weight was observed on lactation Days 4, 7 and 21 at 250 ppm; and throughout lactation at 1250 ppm.
Sex-wise analysis indicated that the mean litter weight of male pups at 250 ppm was significantly reduced on lactation Days 4, 7 and 21 and throughout lactation at 1250 ppm.
Mean female pup weight on lactation Day 7 was found to be increased at 50 ppm only. A significant reduction of female pup weight was observed on lactation Day 4 at 250 ppm. At 1250 ppm, significant decreases in mean body weight of female pups were observed on lactation Days 0, 4, 7, 14 and 21. Hence, the observed mean body weight reduction was considered as treatment related effect at 250 and 1250 ppm.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In pups, the number of days for eye opening was significantly higher at 1250 ppm dose group when compared to control group. The changes were observed in number of days were minimal in numbers and the values were within normal biological variation.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

In male pups, the number of days for balanopreputial separation was significantly higher at 1250 ppm dose group when compared to the control group. The changes observed in number of days were minimal in number and the values were within the normal biological biological variation. The mean body weight at balanopreputial separation day of treatment groups was comparable with current control group.
In female pups, the number of days for vaginal opening of treated groups was comparable with concurrent control group. The mean body weight at vaginal opening day of treatment groups was comparable with concurrent control group.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no effects of treatment in male or female pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross pathological observation did not reveal any treatment related changes.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Findings in F2 offspring were limited to congestion of the spleen observed in one female at 1250 ppm.

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Eye opening was delayed in females at 250 ppm and in both sexes at 250 and 1250 ppm; findings at 1250 ppm were associated with bodyweight deficits.

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

250 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

DISCUSSION:

It is evident from summary tables that both in parental and first filial generation varying degree of pneumonic changes were observed in control and different treated groups almost at comparable level of occurrence. The histopathological features were suggestive of mycoplasmosis. Mycoplasma sp. is ubiquitous in nature and is transmitted by intrauterine route or by aerosol between cage mates including dams to offspring's and between adjacent cages (Anon, 1991). Boorman and Eustis (1990) opined that it is of common occurrence among conventionally reared rats / mice colony, however, rarely complicates toxicological studies.

Varying degree of congestion, deposition of haemosiderin pigments and extramedullary haematopoiesis were recorded in the spleen of animals belonging to different parental and first filial generation groups. The occurrence was higher in treated groups as compared to control both in parental and first filial generation. Almost similar findings were recorded in a sub-chronic (13 weeks) desmedipham toxicity study in rats where the dose rate was higher. (0,300,1200,4800 ppm in the diet) than the present study. (Anon, 1996). The present and earlier findings indicate the haemolytic effect of test substance desmedipham. Congestion and extramedullary haematopoiesis might be its associated effects and supported the findings of Stefanski et al. (1990)

Lesions in reproductive and other organs were either at comparable level both in control and treated groups or were noted in few animals only. Further, these findings were in conformity with our Historical Control Data hence were considered to be spontaneous/ incidental in nature, unrelated to test substance.

Ovarian follicular quantitation in control and high dose group (1250 ppm) of first filial generation did not show any significant difference between these groups indicating that the test substance did not produce any adverse effect on various developmental stages of different types of ovarian follicles (primordial, growing, and antral). Thus, the present study re-supports haemolytic nature of desmedipham, without affecting reproductive cycle in either sex in two generation study.

No treatment related clinical symptoms were recorded in the experimental rats up to the dose level of 1250 ppm in both parental and first filial generation.

No treatment related mortalities were observed during the study period.

Body weight of experimental male rats were not affected by treatment up to the dose level of 1250 ppm.

Treatment related significant reduction in female body weight was observed at 1250 ppm during premating, gestation, and lactation period of both parental and first filial generation.

Feed consumption was not affected by treatment during premating, gestation, and lactation periods of parental and first filial generation.

No major impact on feed efficiency was observed during the experimental period.

All groups female rats were exhibited normal oestrous cycle length during three weeks observation period, prior to cohabitation.

Male fertility, female fertility, gestation, parturition, lactation index, and pregnancy rate were comparable between the control and treated groups in both generations.

No treatment related effect was observed on mean number of live pups, sex ratio, pups survival index, live birth index and pup mortality up to the dose level of 1250 ppm in both generations.

Litter size was not affected by the treatment in both generations. Significant reduction in litter weight of pups (male and female) were observed during lactation period at 250 and 1250 ppm in first filial generation.

The changes observed in the pups for number of days for unfolding of pinna, tooth eruption, eye opening, ear opening, hair growth, vaginal opening and balanopreputial separation were not treatment related and biologically not significant.

Sensory reactivity of pups to stimuli observed for auditory startle, pupil response, air righting reflex and tail pinch response Were considered as not treatment related.

Significant increase in the total motor activity, ambulatory and stereotypic activity were observed in 50,250 and 1250 ppm dose groups of first filial male pups and 250 and 1250 ppm dose groups in second filial, female pups during 0-5 and 5-10 minutes of observation period.

Significant reduction in percent motile sperm and number of epididymal sperm count was observed at 1250 ppm dose level in both generation male rats. Significant reduction in testicular sperm head count was observed in all treated dose level at parental generation. However, male fertility index of parental and first filial generation was not affected during the experimental period.

No treatment related serious changes were observed in rats of both generations in sperm morphological observation up to the dose level of 1250 ppm.

Significant reduction in terminal body weight was observed at 250 and 1250 ppm parental male rats and 1250 ppm first filial generation female rats.

Significant reduction in absolute and relative liver weight of male rat at 250 and 1250 ppm and increase in female rat uterus weight at 50, 250 and 1250 ppm of parental animals were observed.

Significant increase in spleen weight was observed at 1250 ppm parental female rat and first filial generation male rats.

Kidney weight was significantly lower in parental male rat at 50, 250 and 1250 ppm and first filial generation male rat at 1250 ppm and female rat at 250 and 1250 ppm.

Terminal body was significantly lower at 1250 ppm in male and female pups of second filial generation. The brain and thymus weight of male pups of second filial generation was significantly lower at 250 and 1250 ppm.

Gross pathological observation did not reveal any treatment related changes in the animals and pups up to the dose level of 1250 ppm. Varying degree of congestion, deposition of haemosiderin pigments and extramedullary haematopoeisis Were observed in the spleen of treated animals belonging to both parental and first filial generation rats. No other treatment related lesions observed in other organ. Ovarian follicles of primordial, growing, and antral count did not show any treatment related effect at the dose level of 1250 ppm.

No specific reproduction toxicity effect was noticed. However, developmental toxicity was noticed as reduction lactational body weight of pups at 250 and 1250 ppm.

Based on the observations in the present study, it is concluded that the 'No observable' adverse effect level (NOAEL) for desmedipham is 50 ppm concentration in diet for parental rats and pups.

See the results' tables attached in "Overall remarks, attachments"

Applicant's summary and conclusion

Conclusions:

A reproductive NOAEL of 50 ppm (~4 mg/kg bw/d) can be determined for this study based on 50 ppm based on effects on on sperm parameters and and decreased sperm count in F1 at 250 and 1250 ppm.  A parental NOAEL of 50 ppm (~4 mg/kg bw/d) can be determined for this study, based on bodyweight effects. An offspring NOAEL of 50 ppm can also be determined, based on increased motor activity, delayed eye opening at 250 and 1250 ppm and delayed puberty onset at 1250 ppm.

Executive summary:

This study was performed to evaluate the possible effects of desmedipham on reproduction and fertility after repeated oral exposure to the substance via the diet over two generations in rats, in agreement with OECD 416.  Desmedipham technical was administered continuously to three groups of rats of 24 males and 24 females per group, via the diet at concentrations of 0, 50, 250 and 1250 ppm to parental male and female (P) animals prior to the mating for 70 days, during the mating, gestation and lactation phase and through the weaning of the first generation (F1) offspring. The control animals received the untreated diet mixed with the vehicle (acetone) alone. All the parents and F1 generation pups which were not selected for next generation were sacrificed on postnatal day 21. Treatment of the F1 animals which were selected for producing the F2 generation continued during their growth into adulthood and then through mating, gestation, lactation and weaning of the F2 generation pups. The F1 animals and F2 generation pups were euthanized on day 21 of the post-partum period.  Each rat was observed twice daily for morbidity and mortality. All visible signs of toxicity were recorded. Individual body weight and cage wise consumption were recorded weekly. Gestation, lactation and pup data were collected for both generations. Histopathology observations were performed for parental animals. Gross pathology observations were performed for all animals and pups.  No treatment-related clinical signs or mortalities were observed during the study. The body weights of the parental and F1generation males were not affected up to and including the dietary concentration of 1250 ppm, whereas in the females a reduction of the body weight of parental and F1 animals was observed at 1250 ppm during pre-mating, gestation and lactation.  There was no effect on the food consumption of the parental and first F1 generation animals during the whole experimental period.  The parameters, fertility, gestation and pregnancy index were not affected up to the highest dietary concentration of 1250 ppm. Also the live birth index, the survival indices and the lactation index were comparable to the controls. Furthermore, the litter size was comparable between the control and the treated groups. Only in the F2 generation animals a slight reduction of the litter weights in the 250 and 1250 ppm group during the lactation period was seen. No treatment-related pup mortality was observed during the experimental period.  The male fertility index of parental and first filial generation was not affected, reductions in percent motile sperm and number of epididymal sperm and testicular sperm head were observed at 250 and 1250 ppm. Treatment-related changes with regard to sperm morphology were observed in rats of both generations.  Puberty onset was delayed in the high dose of males and females. Motor activity was increased in a dose dependent way in F2 females.  At the end of the study slight reductions of the terminal body weights were mainly observed at 1250 ppm in male rats of the parental generation and in 1250 ppm-females of the F1 generation. Terminal body weight was significantly lower at 1250 ppm in male and female pups of the F2 generation. Organ weight changes seen in some organs, like liver, spleen, uterus and kidneys which were often not dose-related were most likely secondary to the body weight changes at these doses.  Related changes of the brain and thymus weights of male pups of the F2 generation were due to the body weight changes as could be seen from the relative organ weights which were not changed.  Gross pathological observation did not reveal any treatment related changes in the animals at any dose level. Histopathologically, varying degree of congestion, deposition of hemosiderin pigments and extramedullary hematopoiesis, mainly at the highest dose, were observed in the spleen of treated animals belonging to both parental and offspring rats. 

A reproductive NOAEL of 50 ppm (~4 mg/kg bw/d) can be determined for this study based on 50 ppm based on effects on on sperm parameters and and decreased sperm count in F1 at 250 and 1250 ppm.  A parental NOAEL of 50 ppm (~4 mg/kg bw/d) can be determined for this study, based on bodyweight effects. An offspring NOAEL of 50 ppm can also be determined, based on increased motor activity, delayed eye opening at 250 and 1250 ppm and delayed puberty onset at 1250 ppm.