Desmedipham

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Two oral multi-generation studies of reproductive toxicity (OECD 416) in the rat are available for desmedipham.  A screening study (OECD 421/422) is therefore not required.

Test method/  species	Result	Assessment	Reference
OECD 416 (2000 draft) - 2-generation reproductive toxicity study in the rat	Parental NOAEL 50 ppm (3 mg/kg bw/d) based on maternal bodyweight effects.  Reproductive NOAEL 50 ppm (4 mg/kg bw/d) based on reduced sperm count.  Developmental NOAEL 50 ppm based on developmental delays.  Effects on sperm count used to justify proposal for classification in Cat 2 for reproductive toxicity.	Key study	Rajendren and Patel (2015)
US EPA 83-4 - 2-generation reproductive toxicity study in the rat	Parental NOAEL 50 ppm (4-6 mg/kg bw/d) based on haematological findings and thyroid follicular hyperplasia.  Reproductive NOAEL 50 ppm (4-6 mg/kg bw/d) based on reduced litter size.  Developmental NOAEL 50 ppm based on bodyweight effects.	Supporting study	Becker (1986)
OECD 416 - 2-generation reproductive toxicity study in the rat	Parental NOAEL 400 ppm (30-50 mg/kg bw/d) based on bodyweight effects and reduced seminal vesicle weights.  Developmental NOAEL 400 ppm based on reduced litter and pup weights.	The study is not valid.  Reproductive effects cannot be reliably assessed due to limited investigation and methodological deficiencies.	Wilson and Barton (1992)

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-05-30 to 1986-07-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to the guidelines for registering pesticides in the US, hazard evaluation (US-EPA 83-4) corresponding to OECD Guideline 416. There were some limitations and deficiencies if the study is compared to the current guideline 416 from 2001, most of which was mentioned in the study summary above. Furthermore, new endpoints like oestrus cyclicity, AGD, assessment of ovary follicle pool and sperm parameters were not determined. The following organs were not weighed: epididymides, prostate, seminal vesicle and thyroid gland. In addition, Histopathology was not carried out on the F0 generation. From the description of the statistics it is not clear how litter was handled in the analysis (and the use of relevant covariates). Formal adjustment for multiple comparisons were not performed.

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

GLP compliance:

yes (incl. QA statement)

Remarks:

According to the report, the study was conducted under GLP with the possible exception of minor items, none of which were considered to affect the validity of the data or the interpretation of the results in the report.

Limit test:

no

Specific details on test material used for the study:

Light beige colored fine powder

Species:

rat

Strain:

Wistar

Remarks:

Han, outbred, SPF

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSES:
The test article was mixed with granulated feed.

Details on mating procedure:

The F0 generation was paired twice, and F1B litters were used to form the basis of the next generation (F1 generation). Following the weaning of F1B litters on day 21 post partum, the pups were reared for a further seven days on the test diet until they were four weeks old.
Representative 26 male and 26 female pups were selected randomly from each litter/dose level to form the F1 parent animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability over periods up to 21 days was tested in the food mixtures prepared in weeks 0, 3, 8, 11, 17 and 22. Content and homogeneity with one exceptional case at monthly intervals during the study.

Duration of treatment / exposure:

The test article, mixed with granulated feed, was administered during an 80-day pre-pairing and also during the mating, gestation and lactation periods for the breeding of F1A and F1B litters/pups.

Following the weaning of F1B litters on day 21 post partum, the pups were reared for a further seven days on the test diet until they were four weeks old.

The test article was administered during a 100-day pre-pairing period and also during pairing, gestation and lactation periods for breeding of the F2A and F2B litters/pups.

Frequency of treatment:

Continuously

Details on study schedule:

Following the weaning of F1B litters on day 21 post partum, the pups were reared for a further seven days on the test diet until they were four weeks old.
Representative 26 male and 26 female pups were selected randomly from each litter/dose level to form the F1parent animals. The test article was administered during a 100-day pre-pairing period and also during pairing, gestation and lactation periods for breeding of the F2A and F2B litters/pups.

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

50 ppm

Dose / conc.:

250 ppm

Dose / conc.:

1 250 ppm

No. of animals per sex per dose:

30/sex/group

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

Mortality rate: The animals were examined for viability and mortality at least twice daily.
Signs and symptoms: The animals were examined twice daily for signs of toxicity and clinical symptoms.

Oestrous cyclicity (parental animals):

The mating data were used to determine if pregnancy was interrupted after mating, the group mean pre-coital time and to detect marked anomalies of the estrus cycle.

Litter observations:

From each dose level and litter (F1A, F1B, F2A, F2B) one male and one female pup was selected for organ weighing and tissues were preserved for possible histopathological examinations.

Two further male and female pups from each litter per group were reared to at least 32 days post-partum to perform “behavioural”/neuromuscular function tests (cliff avoidance and palmar grasp ability tests and exploratory pattern). The pups selected for behavioural tests and all remaining pups were examined macroscopically.

Postmortem examinations (parental animals):

Shortly after the F1B pups were weaned all F0 parent animals were sacrificed and examined macroscopically.

Postmortem examinations (offspring):

All high-dose and control F1 animals were microscopically examined as well as one male and one female pup from F2B litters in control and high-dose groups (1250 ppm). Following organs were examined; adrenals, bone marrow, eyes, kidneys, liver, lung, lymph nodes, mammary glands, ovaries, pancreas, pituitary, prostate, seminal vesicles, skin, spleen, testes, epididymides, thymus, thyroids and urinary bladder. Also, liver, spleen, thyroid and bone marrow from F1 generation at 50 and 250 ppm were examined in addition.

From each dose level and litter (F1A, F1B, F2A, F2B) one male and one female pup was selected for organ weighing and tissues were preserved for possible histopathological examinations.

Statistics:

Body weights, food consumption, organ weights and ratios, and reproduction data (total number of pups) were evaluated by statistical analysis (Univariate one-way analysis of variance, Student’s t-test, Chi-square test, Wilcoxon Ranks test, Kruskall-Wallis test). Only statistically significant results of organ weights analysed by Student’s t-test were presented in numerical form in the summary tables.

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of males (5-6%) at 1250 ppm were significantly reduced.

The body weight of F0 males were reduced from week 4 to the end of the pre-pairing period and after pairing for F1A litters (days 0 to 56) and F1B litters (days 1 to 22) at 1250 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examinations (gonads, accessory sex glands, gross lesions) were performed only from three P0 males, which failed to induce pregnancy during the second mating period at 1250 ppm. No treatment related pathomorphologic findings were observed in these males.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related effects on mating performance, fertility, mean precoital time, percentage mating, fertility index, conception rate or gestation index were observed during the study.

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: 4 and 6 mg/kg body weight/day for males and females, respectively

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of both sexes of P1 animals at 1250 ppm were significantly reduced (males up to 6% and females up to about 12%).

The body weights of P1 females were reduced throughout the experiment and in P1 males from day 1 to day 22 of the pre-pairing period for breeding of F2A litters at 1250 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption values in F1 parents (males 10-13%, females 13-18%) at 1250 ppm were reduced in a statistically significant manner.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative spleen weights were increased in all groups of P1 animals, statistically significantly in P1 males and females at 250 (males:11 abs, 11%, females: 11%, 14% rel) and 1250 ppm (females: abs 49-54%, rel 50%) (males 47-49% abs, rel 53-55%).

Kidney weights were reduced in P1 females at 1250 ppm (11%,).

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related effects on mating performance, fertility, mean precoital time, percentage mating, fertility index, conception rate or gestation index were observed during the study.

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: 4 and 6 mg/kg body weight/day for males and females, respectively

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant reductions in the body weights of pups were noted in F1A, F1B (10-12%) at 1250 ppm. According to the results, statistically significant reductions in the mean body weights of pups were seen also in F1A litters (5%) at 250 ppm at the end of the lactation period.

Test article-related significantly reduced mean body weights mere noted in group 4 (1250 ppm) pups of the F1A litters from day 1, of the F1B litters from day 7 (5-12%) until day 21 post-partum, respectively.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Brain weights (abs) were reduced in F1A females at 250 (3%) and 1250 ppm (5%), and in F1A and F1B females at 1250 ppm (5%).

In F1A litters and F1B females, the brain weight to body weight ratio was increased at 1250 ppm. Heart weights were reduced in F1A males at 250 (9%) and 1250 ppm (14%).

Spleen weights were reduced in F1B litters at 1250 ppm (16-21% absolute) and in F1A males 1250 ppm (16%, absolute), which is in contradiction to results observed in parent animals where spleen weights were increased at 250 and 1250 ppm.

Thymus weights were reduced in F1A litters in males at 1250 ppm (15%) and in females at 250 (13%) and 1250 ppm (21%). Reductions in weights of testes (9-10% absolute), and adrenals (11-15%) were noted F1A, F1B litters at 1250 ppm.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the F1 generation, treatment related findings were observed in the liver, spleen, bone marrow and thyroid glands. In the liver, minimal or slight hemosiderosis and minimal or slight extramedullary hematopoiesis was observed in both sexes at 1250 ppm. In the spleen, increased hematopoiesis, predominantly erythropoiesis, was observed at a higher incidence and severity in treated parent animals than in controls. Similarly slight hemosiderosis was observed in both control and test animals at all dose levels and moderate hemosiderosis mainly at 250 ppm and 1250 ppm. In the bone marrow slight erythroid hyperplasia was observed in few animals in all treated groups. The findings in liver, spleen and bone marrow were regarded as reactive or compensatory morphologic changes in response to the hemolytic anemia caused by desmedipham.

Summing up cases of slight and moderate follicular hyperplasia in thyroid glands, a dose related increase was observed. The follicular hyperplasia is regarded as a morphokinetic change indicating an altered functional state of the thyroid gland. One F1 male had follicular adenoma at 1250 ppm.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Remarks on result:

other: 4 and 6 mg/kg body weight/day for males and females, respectively

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other: 4 and 6 mg/kg body weight/day for males and females, respectively

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant reductions in the body weights of pups were noted in F2A litters (10-12%) and in F2B litters (7%) at 1250 ppm.

Test article-related significantly reduced mean body weights mere noted in group 4 (1250 ppm) pups of the F2A litters from day 4 (5-12%), and from the F2B litters from day 1 until day 21 post-partum (5-7%), respectively.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Brain weights (abs) were reduced F2A litters at 250 (3-5%).

In F2A litters, the brain weight to body weight ratio was increased at 1250 ppm.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related pathomorphologic findings were observed in the F2B offspring generation.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related pathomorphologic findings were observed in the F2B offspring generation.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Borderline statistical evidence for a reduction of the number of pups in F2A litters at 1250 ppm and in F2B litters at 250 ppm and 1250 ppm was recorded

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Remarks on result:

other: 4 and 6 mg/kg body weight/day for males and females, respectively

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other: 4 and 6 mg/kg body weight/day for males and females, respectively

Reproductive effects observed:

yes

Lowest effective dose / conc.:

250 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

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

Conclusions:

Under the conditions of this study:

Parental NOAEL : 50 ppm (4 and 6 mg/kg body weight/day for males and females, respectively) hemolytic anemia, increases in splenic weights, erythropoiesis or hemosiderosis in spleen and erythroid hyperplasia in bone marrow and follicular hyperplasia in thyroid.

Reproductive NOAEL = 50 ppm (4 and 6 mg/kg body weight/day for males and females, respectively) based on reduced litter size.

Developmental NOAEL = 4 and 6 mg/kg body weight/day for males and females, respectively. Based on reduced body weight and organ weight (kidney, heart, brain).

Executive summary:

To assess its reproductive and developmental toxicity, desmedipham was administered to four groups of 30 male and 30 female Wistar rats (Han, outbred, SPF) at dose levels of 0, 50, 250, or 1250 ppm. The mean intakes of technical desmedipham were approximately 0, 4, 20 and 90 mg/kg bw/day for males and 0, 6, 30, and 140 mg/kg bw/day for females. The test article, mixed with granulated feed, was administered during an 80-day pre-pairing and also during the mating, gestation and lactation periods for the breeding of F1A and F1B litters/pups. The F0 generation was paired twice, and F1B litters were used to form the basis of the next generation (F1 generation). Following the weaning of F1B litters on day 21 post partum, the pups were reared for a further seven days on the test diet until they were four weeks old.
Representative 26 male and 26 female pups were selected randomly from each litter/dose level to form the F1 parent animals. The test article was administered during a 100-day pre-pairing period and also during pairing, gestation and lactation periods for breeding of the F2A and F2B litters/pups.

 

Parental body weight and food consumption were reduced at 1250 ppm. Hemolytic anemia accompanied by significant increases in splenic weights, erythropoiesis or hemosiderosis in spleen and erythroid hyperplasia in bone marrow and follicular hyperplasia in thyroid were observed at 250 and 1250 ppm. Thus the parental toxicity NOAEL was 50 ppm, corresponding to 4 and 6 mg/kg bw/day for males and females, respectively.

 

Significant reductions in body weights of pups were noted in F1A litters at 250 and 1250 ppm. Brain weights were reduced in F1A females at 250 and 1250 ppm. Heart weights, as well as their relative to brain weight ratios, were reduced in F1A males at 250 and 1250 ppm. Relative (to body weight) liver and kidney weights of F2A litters were significantly decreased at 50, 250 and 1250 ppm. Thymus weights, as well as their relative to body weight and brain weight ratios, of F1A females were significantly decreased at 250 and 1250 ppm. The biological relevance of these results, especially concerning organ to body weight ratios at concentrations below 1250 ppm, remain unclear. Besides these observations and other contradictory findings at all doses, significant changes in body or organ weights were found predominantly in litters exposed to the highest dose. Based on the reductions in body and organ weights, the developmental toxicity NOAEL was 50 ppm, approximately of 4 and 6 mg/kg body weight/day for males and females, respectively.

Reference 2

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

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

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 %

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.

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

50 ppm

Dose / conc.:

250 ppm

Dose / conc.:

1 250 ppm

No. of animals per sex per dose:

24/sex/group

Control animals:

yes

Positive control:

Not required

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.

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: 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.

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

Key result

Critical effects observed:

no

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: 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.

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

Key result

Critical effects observed:

no

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.

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:

not examined

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.

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

Key result

Critical effects observed:

no

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

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"

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

4 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A guideline (OECD 416) and GLP-compliant (K1) study is supported by a second (K2) guideline-comparable and GLP-compliant study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The available reproductive toxicity studies have recently (2019) been assessed by RAC; the outcome of these assessments is summarised below.

Key study: 2003

Parental toxicity at the top dose level of 1250 ppm (~140/190 mg/kg bw/d) was limited to modest body weight reductions (up to 9% and 13% in P and F₁ parental females, respectively) and indications of haematotoxicity (increased hemosiderin pigment in the spleen, increased splenic haematopoiesis).  The rationale for the top dose selection was not provided in the study report. RAC noted that desmedipham dose levels of ~4000 ppm induce significant haematotoxicity (Hb reduction by ~15%, MetHb levels of ~10%, pallor) and marked body weight reduction (~20%) in 90-day rat studies.  Therefore, the top dose selection was considered acceptable.  The study reported two findings potentially relevant for fertility classification:

(1) reduced epididymal sperm count in P and F₁ males

Data on sperm parameters showed a statistically significant reduction in epididymal sperm count in both generations at 1250 ppm (~10%) and in the F₁ generation at 250 ppm (~8%).  Testicular sperm count was not appreciably reduced nor was there a biologically significant effect on sperm motility or morphology.  A reduction in epididymal sperm count may generally indicate adverse changes (e.g. reduced epididymal sperm transit time leading to impaired sperm maturation).  However, the concern was reduced by the relatively low magnitude of the decrease, the fact that the reduction was smaller than the difference between generations (i.e. the difference is still within normal variability) and by the lack of biologically
significant changes in other sperm parameters (motility, morphology, testicular sperm count) or in male reproductive organs (histopathology, weight).

(2) delayed puberty onset

Preputial separation (PS) and vaginal opening (VO) were delayed by 2.2 and 2.5 days respectively at the top dose level of 1250 ppm.  Anogenital distance was not measured in this study.  The concern was somewhat reduced by the lack of statistical significance and lack of a dose-response
relationship for the day of vaginal opening and the magnitude of the delay in preputial separation being at the border of normal variability for this endpoint.

Supporting study: 1986

Two litters per generation were produced in this study, with the F₁B litter being selected to form F1 parents. Pup survival data are of limited reliability due to several dams cannibalising their pups.  Parental toxicity at the top dose of 1250 ppm (~90/140 mg/kg bw/d) consisted of
modest reductions in body weight (in lactating F₁ females by ~10 %) and food consumption, increased spleen weight (~50 % in both sexes and generations), increased erythropoiesis and haemosiderosis in the spleen, haemosiderosis in the liver and thyroid follicular hyperplasia.  The choice of the top dose was based on a preliminary experiment.  A slightly reduced litter size at birth was observed in the F₁/F_2B generation at 250 and 1250 ppm.  It is not possible to determine whether the reduction is due to pre- or post-implantation loss from this study.  The effect was not considered by RAC to be of sufficient magnitude to warrant classification.  No other effects related to fertility were identified in this study

Effects on developmental toxicity

Description of key information

A number of studies of oral developmental toxicity (OECD 414) are available for desmedipham, in the rat and rabbit.  A dermal study is also available in the rat.

Test method/ species

Result

Assessment

Reference

OECD 414 (1981) - Rat PNDT study; dose levels 0, 10, 100, 500 mg/kg bw/d: dosing GD 6-15 (24/group).	Maternal NOAEL: 100 mg/kg bw/d (although bodyweight effects were also noted at this dose level)  Developmental NOAEL: 10 mg/kg bw/d based on increased incidences of delayed fetal skeletal ossification, increased incidence of infarct of the liver at 100 and 500 mg/kg bw/d	Supporting study	Jacob (2001)
OECD 414 - Rat PNDT study; dose levels 0, 10, 100, 1000 mg/kg bw/d: dosing GD 6-15 (25/group).	Maternal NOAEL: 10 mg/kg bw/d based on bodyweight effects.   Developmental NOAEL: 10 mg/kg bw/d based on increased incidence of supernumerary rib.  Agnathia/micrognathia at high dose levels used to support classification in Cat 2.	Key study	Becker et al (1985)  embryotoxicity
OECD 414 - Rat PNDT study; dose levels 0, 10, 100, 500 mg/kg bw/d: dosing GD 6-15 (35/group).	Maternal NOAEL: 10 (7) mg/kg bw/d based on bodyweight effects   Developmental NOAEL: 100 (70) mg/kg bw/d based on reduced fetal weight and reduced ossification	Key study	Becker et al (1985), teratogenicity
Non-guideline - Rat: RF study performed at 0, 125, 250, 500, 1000 mg/kg bw/d: dosing GD 6-15 (8-9/group)	Maternal NOAEL: 500 mg/kg bw/d based on reduced bodyweights and food consumption   Developmental NOAEL: 250 mg/kg bw/d based on reduced fetal weights.	This range-finding study is not considered to be valid due to the small group size and as skeletal and visceral findings were not investigated	Barton (1990)
OECD 414 - Rat PNDT study; dose levels 0, 60, 250, 1000 mg/kg bw/d: dosing GD 6-16 (25/group).	Maternal NOAEL: 60 mg/kg bw/d based on reduced weight gain and food consumption; clinical signs and increased spleen weight  Developmental NOAEL: 250 mg/kg bw/d based on reduced fetal weight.¿ Three fetuses from one litter at 1000 mg/kg bw/d had cleft palate. Findings used to support classification in Cat 2.	Supporting study	Barton (1991)
OECD 414 - Rat dermal PNDT study; dose levels 0, 1000 mg/kg bw/d: dosing GD 6-15 (25/group).	There were no effects of treatment in this dermal study: the maternal and developmental NOAEL values are therefore 1000 mg/kg bw/d	Supporting study	Barton (1988)
Non-guideline - Summary of historical control data	Not specified.	Not relevant: not a study	Mallyon (2001)
OECD 414 - Rabbit PNDT study; dose levels 0, 50, 150, 450 mg/kg bw/d: dosing GD 6-27 (16/group).	Maternal NOAEL: <50 mg/kg bw/d based on reduced weight gain and food consumption   Developmental NOAEL: <50 mg/kg bw/d based on increased post-implantation loss and reduced fetal weight	Supporting study	Becker et al (1984)
Non-guideline - Rabbit: RF study performed at 0, 40, 120 and 360 mg/kg bw/d: dosing GD 6-18 (8/group)	Maternal NOAEL: <40 mg/kg bw/d based on reduced bodyweight   Developmental NOAEL: 120 mg/kg bw/d based on reduced fetal weight, increased post-implantation loss.	This range-finding study is not considered to be valid due to the small group size and as skeletal and visceral findings were not investigated	McCay and Barton (1991)
OECD 414 - Rabbit PNDT study; dose levels 0, 30, 90, 270 mg/kg bw/d: dosing GD 6-18 (16/group).	Maternal NOAEL: 30 mg/kg bw/d based on increased spleen weight  Developmental NOAEL: 30 mg/kg bw/d based on increased early embryonic death; increased incidence of caudal pelvic shift  The number of dams with litters was low across all groups (12-15), limiting the sensitivity of this study	Key study	McCay and Barton (1991)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2000-07-24 to 2001-05-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

See Principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

The study was performed after guideline 414, "Teratogenicity" (Adopted on May 12, 1981).
After the study was performed, a new version of the OECD Test Guideline 414 has been adopted 22nd January, 2001. Deviation from new guideline: dosing from GD6-15 instead of GD 5 to day prior to caesarean section (day 20). Body weight and food consumption was determined every 5 days and not every 3 days as the current guideline dictates.
It is not described in the statistics if covariates were considered.

GLP compliance:

yes (incl. QA statement)

Remarks:

A GLP study audit was requested for this study as it was conducted by the same laboratory as the two-generation rat study (Rajendren, 2003) where a study audit was required based on the doubts about the quality of the reporting of the study.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Weight at dosing: Females: 150-300 g
Acclimation period: 7 days
Diet: Ground diet
Water: drinking water ad libitum in polypropylene bottles
(capacity 300 ml) filtered through aquaguard water filtration system.
Housing: Solid floor Polypropylene cages (410 x 282 x 180 mm) on a rack.
Environmental conditions
Temperature: 20-24 °C
Humidity: 65 ± 2.88 %
Air changes: 20-25
Photoperiod: 12 hours

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Aqueous 0.5 % CMC (vehicle) Carboxy methyl cellulose sodium salt

Details on exposure:

Duration of dosing:
The dams received desmedipham technical for 10 consecutive days, starting from day 6 to 15 of gestation (organogenesis period) at dose levels of 10, 100 and 500 mg/kg bw. The control group received aqueous 0.5% CMC only.
Dose preparation and stability:
Dosing suspensions were prepared freshly daily at the same time and were dosed during the morning hours. The specified amount of desmedipham was calculated and weighed for all groups. The required volume of aqueous 0.5 % CMC (vehicle) with desmedipham technical was first homogenized using mortar and pestle and added into the beaker and then mixed using a magnetic stirrer for approximately thirty minutes. The appropriate volume of dosing suspension for each group was then allocated into beakers in a serial dilution pattern at the concentrations of 50 mg/ml, 10 mg/ml and 1 mg/ml. Actual concentration of desmedipham varied within 93.3% of nominal concentration according to analysis.
Cohabitation:
After acclimatization male and female rats were cohabited until the required numbers of animals were obtained depending on the oestrus cyclicity of the female rats. Mating was detected by observation of spermatozoa in vaginal smear. The females were examined each morning for evidence of mating by examination of the vaginal smear for spermatozoa. Females showing spermatozoa were separated and referred as “dam”. The day of identification of spermatozoa was termed as Day “0” of gestation. The total number of dams obtained in the study were 24 in each group.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

9 days

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: See Dose Level Selection Criteria.
In-life dates: Start: September 7, 2000 End: January 25, 2001
Animal assignment and treatment:
A total of 120 females and 40 males were initially used for the study.
Methods
Observations:
During the study period the details of clinical signs of toxicity including changes in skin and fur, eye and mucous membrane, respiratory, circulatory, autonomous and central nervous system and uro- genital system of the dams were carefully noted and recorded daily.
Body weights and food consumption:
The body weights and the amount of feed left over by each of the pregnant dams was recorded every five days during gestation days i.e., on day 0, 5, 10, 15 and 20 of gestation.
Necropsy:
At the 20th day of gestation, the female animals were humanely sacrificed using an overdose of carbon dioxide. Immediately the maternal viscera including uteri were examined macroscopically and pathological were recorded. The gravid uterus was removed without separating the left and right horns, excess fat was trimmed off, blotted free of blood using absorbent filter paper and weighed. The ovaries were removed and the corpora lutea were counted immediately. The uteri of any animal judged to be non-pregnant was stained with 5% aq (v/v) ammonium sulphide solution and examined for implantation sites (Salewski 1964).

Fetal examinations:

The fetuses were examined for gross external anomalies using a magnifying hand lens. The abnormal fetuses were recorded, preserved and stores as per their respective groups. After examination in each litter, the abdominal and thoracic cavity of approximately 50% of fetuses were dissected and carefully examined for visceral anomalies (Wilson, 1965). After completing the visceral examination, the fetuses were stored in aceto-alcohol-formalin and the cephalic region of the fetuses were subjected to razor sectioning in order to visualize internal structures of the head including the symmetry of external nares, nasal conchae, nasal septum, palate, the development of the cerebellum and brain stem. Five transverse sections of the fetal cephalic regions were observed in Labomed sterozoom microscope. The deviations from the normal development of the cephalic region were recorded. In the skeletal examination (Dawson, 1926), the remaining fetuses (50%) of each individual litter were processed using Alizarin Red S rapid single staining technique for a week duration after fixing in 70% isopropyl alcohol for one day. Then the fetuses were stained in alizarin red solution (0.0025%) for one day, macerated with 1% aqueous potassium hydroxide (KOH) solution for approximately one day and then placed in clearing and hardening fresh solution of glycerol, 70% isopropyl alcohol and benzyl alcohol (2:2:1) for one day and finally stored in a mixture of (1:1) of 70% isopropyl alcohol and glycerol (Gurr, 1956). After 4 hours of preservation in 1:1 mixture, the stained fetuses were examined under Labomed Stereoscopic zoom microscope at a magnification of 12.5 X and above. The skull was examined for size, shape and degree of ossification of nasal, frontal, parietal, interparietal, occipital, lacrimal, zygomatic, squamosal, pre-maxillary, mandible, basisphenoid, exoccipital and tympanic bullae. Similarly, the vertebral arches and its centres (of thoracic, lumbar and sacral), ribs and sterna centre were also examined for size, shape and counted for the number of ossification centres. The thoracic, pelvic girdles, fore limbs and hind limbs were examined for the development of flat, long bones and in phalanges – the number of metacarpal and metatarsals. Any deviation from the normal development of the above mentioned bones was recorded for each foetus.

Statistics:

Raw data were processed to give group means and standard deviations with significance wherever the difference occurs between the control and treated groups using suitable statistical techniques (e.g. Student's 't' test) for body weight gain, feed consumption, litter data, prenatal data etc.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No difference in the body weight data of control and treated groups was seen. Gestational body weights, 20-day dam corrected body weights (13%, 13%, 14% ,14 % in control, 10, 100 and 500 mg/kg bw/day, respectively) and feed consumption in the treated groups were comparable to control group throughout the gestation period. Body weight gain was slightly lower at gestations days 6-10 (15%) and 11-15 (5%) at 500 mg/kg bw/day, however, in the absence of effect on absolute body weight, the effect on body weight gain was not considered adverse.

Other effects:

no effects observed

Description (incidence and severity):

Necropsy findings: No treatment-related findings were seen.

Details on results:

No treatment-related clinical signs or mortality were observed during the study. No difference in the pregnancy data of control and treated groups was seen. Prenatal parameters, number of corpora lutea, implantation rates, live and dead foetuses and resorptions were not affected by treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

effects observed, treatment-related

Localisation:

other: body weight

Description (incidence and severity):

dose-dependent decrease in the body weight gain observed in the high dose of 500 mg/kg bw per day.

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No significant difference was observed in fetal external observation data up to a dose level of 500 mg/kg bw.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Fetal skeletal observations of ossification variations revealed statistically significant difference in incomplete ossification of interparietal bone of fetal and litter incidence at 500 mg/kg bw and of fetal incidence of bipartite occification in sternebrae at 100 and 500 mg/kg bw/day (Table: Summary of skeletal findings). Fetal skeletal observations of skeletal deviations revealed no significant change in fetal and litter incidence at dose level of 500 mg/kg body weight.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Fetal visceral observations revealed significant difference in the fetal incidence of infarct of liver at the dose level of 100 and 500 mg/kg body weight. Fetal visceral observations revealed significant difference in fetal and litter incidence in haemorrhagic kidney at all doses.

Other effects:

no effects observed

Description (incidence and severity):

Fetal head-razor section observations revealed no significant difference at a dose level of 500 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:

Litter Data: The mean litter size, litter weights, average fetal weights and mean number of male and females per litter were not affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: sternum

visceral/soft tissue: hepatobiliary

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Table: Summary of body weight gain compared with control (%) data in dams

% of control	Groups (no observation)
Gestation periods	G2 (20)	G3 (22)	G4 (22)
0-5	16.0 %	21.1 %	12.6 %
6-10	11.6 %	-4.5 %	-15.0 %
11-15	-13.5 %	18.1 %	-4.7 %
16-20	0.0 %	-1.5 %	10.0 %

Conclusions:

A maternal NOAEL of 100 mg/kg bw/d dose-dependent was determined for this study based on a decrease in the body weight gain observed in the high dose of 500 mg/kg bw per day.
A developmental NOAEL of 10 mg/kg bw/d was determined for this study based on an increased incidence of infarct of liver, bipartite ossification of sternebra at 100 and 500 mg/kg bw/day. Increased incidence incomplete ossification of interparietal bone at 500 mg/kg bw/day.

Executive summary:

The Study was performed to evaluate the possible teratogenic potential of desmedipham technical by oral route in pregnant Wister rats. The methods followed were as per the guideline of: The organization of Economic Co-operation and Development (OECD) N° 414, adopted on May 12, 1981, entitled “Teratogenicity”. Pregnant Wistar rats were exposed daily to desmedipham technical by oral intubation at dose level of 0, 10, 100 and 500 mg/kg bw, during gestation days (GD) 6 to 15 using 0.5% CMC as vehicle. Concurrent control group of pregnant rats were administered aqueous 0.5% CMC orally. Maternal body weights, clinical observations and feed consumption were recorded throughout the gestation period. At the scheduled euthanization, on GD 20, the uteri and ovaries were removed, weighed and examined for the number of corpora lutea, implantation sites, resorption sites and for live and dead fetuses. The fetuses were weighed and examined for external, visceral and skeletal observations. No mortality was observed in the control and the treated groups. Pregnancy rate did not show any significant change up to the dose levels of 500 mg/kg bw. Macroscopically no major treatment related effects was observed pathologically up to the dose level of 500 mg/kg bw. Gestational body weights, 20-day dam corrected body weights and feed consumption in the treated groups were comparable to control group throughout the gestation period. Percent body weight gain although slightly reduced from GD 6- 15, did not reveal any sign of significant changes during the gestation period up to the dose level of 500 mg/kg bw. From the studies with repeated administration hematological effects at the highest dose can be assumed. Percent relative uterine weights did not reveal any significant change up to the high dose group of 500 mg/kg bw. The parameters in prenatal data such as pre/post implantation loss and sex ratio did not have significant changes up to a dose level of 500 mg/kg bw. No significant change was observed in the fetal body weights and litter weights at 500 mg/kg bw. No significant difference was observed in the litter size, litter weight in the treated groups as compared to the control. No significant difference was observed in fetal external observation data up to a dose level of 500 mg/kg bw. Fetal visceral observations revealed significant difference in the fetal incidence of infarct of liver at the dose level of 100 and 500 mg/kg bw. Fetal head-razor section observations revealed no significant difference at a dose level of 500 mg/kg bw. Fetal skeletal observations of ossification variations revealed significant difference in incomplete ossification of interparietal bone at 500 mg/kg bw/day and bipartite occification in sternebrae of fetal incidence at 100 and 500 mg/kg bw. Fetal skeletal observations of skeletal deviations revealed no significant change in fetal and litter incidence at dose level of 500 mg/kg bw. It is concluded that the No-Observed Adverse Effect Level (NOAELfoetotoxicity is 10 mg/kg bw. Maternal NOAEL is 100 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991-01-17 to 1991-11-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

An OECD guideline was not mentioned in the study report but, according to earlier assessment, the study complied with the older version of guideline 414 with the above mentioned limitations. After the study was performed, a new version of the OECD Test Guideline 414 has been adopted 22nd January, 2001. The main study fulfils these data requirements except that body weights were recorded on Days 0, 6, 9, 13, 17 and 20 of gestations instead of every 3 days. Dosing period was from gestation day 6 through to day 15 and not from day 5 post mating to the day before caesarean section. Statistics were very briefly described and the use of covariates were only mentioned in the analysis of spleen weight.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

2% carboxymethylcellulose at a dose volume of 10 ml/kg bw.

Details on exposure:

The test article was dosed orally by gavage as a suspension of 2% carboxymethylcellulose at a dose volume of 10 ml/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of dosing suspensions conducted on the first day of dosing indicated that all suspensions were 8 - 15% below the nominal level. The high dose suspension was analysed on the third day of dosing and all groups were analysed on the ninth day of dosing. All these suspensions were analysed and were shown to be within ±5% of the nominal level.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were agreed with the Sponsor after evaluation of the results of a separate dose range finding study (IRI Project No. 490070) in which maternal toxicity, indicated by reduced weight gain and food consumption, was seen at 1000 mg Desmedipham.kg^-1.day^-1.
- Rationale for animal assignment (if not random): random.
- Fasting period before blood sampling for (rat) dam thyroid hormones: n/a
- Time of day for (rat) dam blood sampling: n/a
- Other:
Desmedipham (Purity 98%) was administered to four groups of 25 mated female rats at dose levels of 0, 60, 250 and 1000 mg/kg bw/day. A batch of 120 sexually mature virgin female Sprague-Dawley; rats were mated with 60 sexually mature males of the same strain. The dams received desmedipham once daily over gestational days of 6 to 16.

Maternal examinations:

Body weight and food consumption were recorded during the study and spleen weight at the termination of the study.

Ovaries and uterine content:

Dams were necropsied on day 20 of gestation. The contents of the thoracic abdominal cavities were examined macroscopically and the reproductive tract was removed, weighed, opened and the contents examined. A number of corpora lutea graviditas in each ovary and the position of all implatation sites in uterus were recorded and classified.

Fetal examinations:

Each live fetus was individually identified, weighed and examined for externally visible abnormalities. Approximately one half of the fetuses from each uterus was fixed in methylated ethyl alcohol, and the remaining half in Bouin’s fluid, for examination of soft tissue abnormalities and variants by the Wilson technique. The fetuses in alcohol were examined for visceral abnormalities by open dissection, the skeletons were stained with Alizarin Red, and the skeletal structures in these fetuses were examined for abnormalities and variants.

Statistics:

Spleen weight was statistically analysed and results were presented in tabular form. The other analysed parameters or statistical test methods used in this study were not reported.

Urinalysis findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Details on results:

One dam with total resorptions (4 embryos) at 250 mg/kg bw/day was excluded from the calculations of body weight, food consumption and reproduction data. One early deceased dam with 15 live implants at 250 mg/kg bw/day was also not included in the calculations. Individual clinical and necropsy findings on each test animal were not reported.

Most animals (18/25) receiving 1000 mg/kg bw/day showed discoloured urine (grey, mild/dark brown or purple/black) on one or more occasions, and three animals (peach or light brown stained urine) at 250 mg/kg bw/day, indicating hemolytic anemia. During the course of the necropsies, enlargement of the spleen was noted at 1000 mg/kg bw/day. The spleen weights of all pregnant animals were therefore recorded. The mean spleen weights were significantly increased in 250 (19 %) and 1000 (81%) mg/kg bw/day dose groups compared with control.

Maternal toxicity was also indicated by reduced food consumption (8%) and body weight gain (29%) at 1000 mg/kg bw/day (Table: Group mean body weight gain (g) ± standard deviation of pregnant animals). Statistically significant results were not reported or marked in the tables of Group mean body weights and Group mean food consumption.

Corrected body weight gains (weight gain GD 6-17-uterus weight) were -10.4 g, -9.5 g, -11.9, -22 g at 0, 60, 250 and 1000 mg/kg bw/day, respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

urinalysis

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: spleen

Description (incidence and severity):

increased spleen weight.

Fetal body weight changes:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

One dam with total resorption (4 embryos) at 250 mg/kg bw/day was excluded from the final evaluation without any explanation.

The number of embryonic resorptions was about 3-fold higher in all treated groups than in the control group (Table: Summary of reproduction parameters, malformations and anomalies). Some fetuses with major visceral or skeletal malformations were observed in the dosed groups displaying an equivocal overall picture of teratogenicity; some of the effects were probably due to maternal toxicity at dose levels above 250 mg/kg bw/day. The total number of implants affected was increased in a dose dependent way in all treated groups. Since the total number of affected implants was mainly influenced by the post-implantation losses, the toxicological value of this parameter is highly limited. The mean fetal weight was 20% lower at 1000 mg/kg bw/day than in the control group.

At 60 mg/kg bw/day one fetus had moderate internal hydrocephaly and bilateral anophtalmia and the other one umbilical hernia. At 250 mg/kg bw/day 7 fetuses from the same litter showed slight kyphosis, shortened ribs and misshapen clavicles. Cleft palate was observed in 3 fetuses and cardiovascular malformations/ abnormalities were recorded in 5 fetuses including one fetus with multiple malformations (interventricular septal detect, hydronephrosis, testes not fully descended) at 1000 mg/kg bw/day. Interventricular septal defect was also observed in two other fetuses and partial duplication of inferior vena in 2 fetuses from the same litter at 1000 mg/kg bw/day. There was an increase in retardation of ossification of occipital bone and the scapulae in fetuses at 1000 mg/kg bw/day.

The testes of 4 fetuses were not fully descended to the pelvic position in the same litter, probably associated with the reduced fetal weight at 1000 mg/kg bw/day. Subcutaneous haemorrhage was observed in two fetuses (upper ventral thorax, nasal region) at 250 mg/kg bw/day and in two fetuses (rights cranial region, dorsal cervical region, right pinna) at 1000 mg/kg bw/day (Table: Summary of reproduction parameters, malformations and anomalies).

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: body weight

Description (incidence and severity):

reduced.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Table: Summary of reproduction parameters, malformations and anomalies

End point	Dose levels (mg/kg bw/day) 0                       60		250	1000
Number of dams	20	24	23 1)	21
Corpora lutea	319	397	373	361
Implantations	304	373	358	351
Embryonic resorptions	9	23	24	27
Fetal resorptions	0	0	0	3*
Postimplantation loss (% of implantations)	9(3.0)	23(6.2)	24(6.7)	30(8.5)
Live fetuses	295	350	334	321
Mean Implantations/dam	15.2	15.5	15.6	16.7
Mean Live fetuses/dam	14.8	14.6	14.5	15.3
Visceral investigations
Number of fetuses examined in Bouins fluid	142	168	162	155
Number of fetuses examined viscerally	295	350	334	321
Moderate internal hydrocephaly		1
and bilateral anophtalmia
Intraventricular septal defect, hydronephrosis Intraventricular septal defect				1* 1a)*
Intraventricular septal defect, right atrium				1
enlarged, reduced right ventricle
Partial duplication of inferior vena cava				2(1)
Umbilical hernia with protrusion of intestine	1	1
Subcutaneous haemorrhage			2(2)	1^a)+1(2)
Hepatic haemorrhage	1	1
Unilateral increased renal pelvic cavitation		2(2)
Skeletal investigations (litters)
Number of fetuses examined skeletally	153	182	172	166
Cleft palate				3(1)
Slight kyphosis of cervico/thoracic spine, dorsal-			7(1)
ventral curvature of sternum^b)
Occipital bone and scapulae retarded/slightly				11(3)
retarded ^2)
Mean litter mean fetal weight (g)	3.76±0.28	3.67±0.20	3.63±0.25	2.99±0.34
% compared to control		(-2.4)	(-3.5)	(-20.5)
Total number of implants affected ^3)(%)	11(3.6)	28(7.5)	33(9.2)	39(11.1)

* In the same litter, (number of litters), a) Intraventricular septal defect, absent innominate artery, misshapen genital papilla, subcutaneous haemorrhage in right cranial region and dorsal cervical region, b) Clavicles misshapen, ribs shortened and thickened bilateral forelimb flexure, 1) One dam with total resorption (4 embryos) excluded from the evaluation, 2) Not included in the evaluation of implants affected, 3) Implants affected = resorptions, major visceral and skeletal malformations/abnormalities (did not include skeletal retardation)

Cleft palate is a rare finding and was observed in 3 fetuses from the same litter at 1000 mg/kg bw/day. Partial duplication of inferior vena cava (2 from 1 litter) was observed as well at 1000 mg/kg bw/day. These effects may be related to treatment or just a common genetic characteristic of the fetuses. Since the cause of these observations cannot be excluded to be related to treatment, these findings cause additional concern for developmental toxicity of desmedipham.

Table: Summary of reproduction parameters and developmental parameters

End point	Dose levels (mg/kg bw/day) 0                        60                       250                        1000
Number of dams	20	24	23 1)	21
Corpora lutea	319	397	373	361
Implantations	304	373	358	351
Embryonic resorptions	9	23	24	27
Fetal resorptions	0	0	0	3*
Postimplantation loss (% of implantations)	9(3.0)	23(6.2)	24(6.7)	30(8.5)
Live fetuses	295	350	334	321
Visceral investigations
Number of fetuses examined in Bouins fluid	142	168	162	155
Number of fetuses examined viscerally	295	350	334	321
Moderate internal hydrocephaly		1
and bilateral anophthalmia
Intraventricular septal defect, hydronephrosis Intraventricular septal defect				1* 1a)*
Intraventricular septal defect, right atrium				1
enlarged, reduced right ventricle
Partial duplication of inferior vena cava				2(1)
Umbilical hernia with protrusion of intestine	1	1
Subcutaneous haemorrhage			2(2)	1^a)+1(2)
Hepatic haemorrhage	1	1
Unilateral increased renal pelvic cavitation		2(2)
Skeletal investigations (litters)
Number of fetuses examined skeletally	153	182	172	166
Cleft palate				3(1)
Slight kyphosis of cervico/thoracic spine, dorsal-			7(1)
ventral curvature of sternum^b)
Occipital bone and scapulae retarded/slightly				11(3)
retarded ^2)
Mean litter mean fetal weight (g)	3.76±0.28	3.67±0.20	3.63±0.25	2.99±0.34
Total number of implants affected ^3)(%)	11(3.6)	28(7.5)	33(9.2)	39(11.1)

* In the same litter, (number of litters), a) Intraventricular septal defect, absent innominate artery, misshapen genital papilla, subcutaneous haemorrhage in right cranial region and dorsal cervical region, b) Clavicles misshapen, ribs shortened and thickened bilateral forelimb flexure, 1) One dam with total resorption (4 embryos) excluded from the evaluation, 2) Not included in the evaluation of implants affected, 3) Implants affected = resorptions, major visceral and skeletal malformations/abnormalities (did not include skeletal retardation)

Postimplantation loss increased from 3.0% in controls to 6.2-8.5% in treated animals, mostly due to embryonic resorptions, but also at the high dose partly due to a few fetal resorptions. Maternal toxicity was observed at the high dose (1000 mg/kg bw/day) by reduced food consumption (-8%) and body weight gain (-29%).

The historical control data in SD rats show that the incidences of postimplantation loss seen in treatment groups of the teratology study were comparable with those seen in historical controls. No evidence of treatment-related increases in postimplantation losses were seen the other two rat teratology studies, and no effects on the reproductive performance in two rat multigeneration studies were observed. The data as a whole would therefore indicate that desmedipham has no effect on reproduction in the rat.

The historical control data in SD rats show that the incidences of postimplantation loss seen in treatment groups of the teratology study by (8.5%) were just above those seen in historical controls (up to 8%). Historical controls were from the period October 1989 to October 1992, from the performing laboratory and considered acceptable by Co-RMS: The full position paper is reproduced below.

Conclusions:

Maternal toxicity was clearly demonstrated by reduced food consumption, body weight gain, discoloration of the urine and by increased spleen weight at 1000 mg/kg bw/day. Since at 250 mg/kg bw/day still some effects could be attributed to the treatment, the maternal toxicity NOAEL was 60 mg/kg bw/day in this study.

The embryotoxic effects of the substance at doses which do not clearly cause maternal toxicity included post-implantation losses which were increased in all treated groups. Since no historical control values were presented for this rat strain and this particular endpoint, the relevance of the post- implementation losses remains unclear. All other affected end-points display equivocal results or can be related to maternal toxicity. Therefore as a conservative approach, the embryotoxicity NOAEL has to be settled lower than 60 mg/kg bw/day.

The study was acceptable despite some deficiencies in statistical analyses.

NOAEL maternal : 60 mg/kg bw/day based on increased spleen weight and discolored urine.

NOAEL developmental : 250 mg/kg bw/day based reduced body weight.

Executive summary:

Desmedipham (Purity 98%) was administered to four groups of 25 mated female rats at dose levels of 0, 60, 250 and 1000 mg/kg bw/day. A batch of 120 sexually mature virgin female Sprague-Dawley; Charles River CD strain (outbred albino) rats were mated with 60 sexually mature males of the same strain. The dams received desmedipham once daily over gestational days of 6 to 16. The test article was dosed orally by gavage as a suspension of 2% carboxymethylcellulose at a dose volume of 10 ml/kg bw. The analyses of dosing suspensions conducted on the first day of dosing indicated that all suspensions were 8 - 15% below the nominal level. The high dose suspension was analysed on the third day of dosing and all groups were analysed on the ninth day of dosing. All these suspensions were analysed and were shown to be within ± 5% of the nominal level. Body weight and food consumption were recorded during the study and spleen weight at the termination of the study. Spleen weight was statistically analysed and results were presented in tabular form. The other analysed parameters or statistical test methods used in this study were not reported.

Dams were necropsied on day 20 of gestation. The contents of the thoracic abdominal cavities were examined macroscopically and the reproductive tract was removed, weighed, opened and the contents examined. A number of corpora lutea graviditas in each ovary and the position of all implatation sites in uterus were recorded and classified. Each live fetus was individually identified, weighed and examined for externally visible abnormalities. Approximately one half of the fetuses from each uterus was fixed in methylated ethyl alcohol, and the remaining half in Bouin’s fluid, for examination of soft tissue abnormalities and variants by the Wilson technique. The fetuses in alcohol were examined for visceral abnormalities by open dissection, the skeletons were stained with Alizarin Red, and the skeletal structures in these fetuses were examined for abnormalities and variants.

NOAEL maternal : 60 mg/kg bw/day based on increased spleen weight and discolored urine.

NOAEL developmental : 250 mg/kg bw/day based reduced body weight.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-01-11 to 1988-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

The study complied with the older version of guideline 414. After the study was performed, a new version of the OECD Test Guideline 414 has been adopted 22nd January, 2001. The main study fulfils these data requirements except that the dosing period was from day 6 through to day 15 post coitum and not from day 5 post mating to the day before caesarean section. Food consumption was recorded with 5 days intervals instead of 3 days.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Route of administration:

dermal

Vehicle:

other: sesame oil

Details on exposure:

A suspension of desmedipham technical in sesame oil was applied dermally occluded 6 hours/day from day 6 through to day 15 post coitum at a single dose level of 1000 mg/kg bw/day at a dose volume of 2.5 ml/kg bw.

Duration of treatment / exposure:

9 days

Frequency of treatment:

6 hours/day for 9 days.

Duration of test:

21 days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were based on the results of the preliminary study (RCC Project 095073).
- Rationale for animal assignment (if not random): Random.
- Fasting period before blood sampling for (rat) dam thyroid hormones: n/a
- Time of day for (rat) dam blood sampling: n/a
- Other: n/a

Maternal examinations:

On day 21 of gestation, dams were necropsied and the fetuses were removed by caesarean section. Post mortem examination included gross macroscopic examination of all internal organs, with emphasis on ovaries (number of corpora lutea), uterus and its contents and the position of fetuses.

Fetal examinations:

The fetuses were removed, sexed, weighed and examined for gross external abnormalities. Approximately one half of the fetuses from each litter were examined for skeletal abnormalities and variants by placing fetuses in solution of potassium hydroxide for clearing and stained with Alizarin red S. The remaining half were examined for soft tissue abnormalities by Wilson’s technique.

Statistics:

Body weights, food consumption and reproduction and skeletal data were analysed statistically (Univariate one-way Analysis of Variance, Student’s t-Test, U-test, Fisher’s Exact test). Statistically significant results were presented in the statistical analysis report, but not in tabular form, except for skeletal findings in fetuses.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in body weights of dams between the treated group and the control group. Body weight gains were slightly reduced on days 11 to 15 of the treatment and corrected body weight gain were 5.4% and 3.8% reduced at 0 and 1000 mg/kg bw/day. No maternal macroscopic abnormalities were detected. See Table: Body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No skin reactions.

Details on results:

There were no deaths, no treatment-related clinical signs, no skin reactions and no effect on food consumption.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

The percentage of embryonic resorptions was 9.9% in the treated group and 7.8% in the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

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

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See visceral.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal investigations revealed one fetus with left ribs wavy with retarded ossification at 1000 mg/kg bw/day.
The incidences of fetuses with non-ossified cervical vertebra 1 and 2 were significantly increased at 1000 mg/kg bw/day (both 26%), compared to the control group (11% and 6%, respectively).
Since the control incidence of fetuses with non-ossified cervical vertebra 2 was outside the historical control range from the conducting laboratory (1984-1986) (13-32%) of the investigating laboratory, the relevance of the finding remains unclear and is probably biased by chance.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were two malformed fetuses (2/305) in the control group and three malformed fetuses (3/300) in the treated group in external and visceral examinations. One runt fetus with anasarca and dilation of the lateral ventricles of brain and one fetus with bilateral malrotated hind limbs was observed in the control group. In the treated group, there was one runt fetus with generally retarded ossification, one fetus with left hind limb malrotated and one fetus with slight dilatation of the lateral ventricles of the brain.

Details on embryotoxic / teratogenic effects:

The numbers of corpora lutea, implantation sites and live fetuses (the incidences of pre and post implantation losses), fetal weights and the sex ratios were not affected by the treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Key result

Abnormalities:

no effects observed

Conclusions:

Corrected body weight gain were 5.4% and 3.8% in control and treated group, respectively, indicating maternal toxicity at 1000 mg/kg bw/day.

NOAEL maternal < 1000 mg/kg based on reduced bw gain
NOAEL developmental > 1000 mg/kg bw/day

Executive summary:

The purpose of this study was to assess the effects of dermal application of DESMEDIPHAM TECHNICAL on embryonic and fetal development in pregnant Wistar/HAN rats. Both groups consisted of 25 mated female rats. DESMEDIPHAM TECHNICAL was applied dermally, occluded, 6 hours/day from day 6 through to day 15 post coitum at a single dose level of 1000 mg/kg body weight/day. The Control group was similarly treated with the vehicle alone (sesame oil). The dosage was based on the results of the preliminary study (RCC Project 095073). A standard dose volume of 2.5 ml/kg body weight with a daily adjustment to the actual body weight was used. Females were sacrificed on day 21 post coitum and the fetuses removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.
The following results were obtained:
- There were no deaths during the study and no systemic clinical
signs or local skin reactions related to treatment with
DESMEDIPHAM TECHNICAL.
- Food consumption was similar in both groups. There was a minimal retardation in body weight gain during the latter half of the dosing period and at termination, overall body weight gain corrected for uterus weight was slightly lower than that of Controls.
- At necropsy, all females were pregnant and had live young. No macroscopic changes were noted in the dams at necropsy.
- Reproduction parameters - numbers of corpora lutea, implantations and live fetuses and incidences of pre- and post-implantation losses - were not affected by treatment with DESMEDIPHAM TECHNICAL.
- The fetal parameters, sex ratios, mean fetal body weights, incidences of external, visceral and skeletal abnormal findings and incidences of skeletal variants were not affected by treatment with DESMEDIPHAM TECHNICAL.

DESMEDIPHAM TECHNICAL was administered by dermal application to mated Wistar/HAN rats at a single dose level of 1000 mg/kg body weight/day from day 6 through to day 15 post coitum in order to assess the effects of the compound on embryonic and fetal development. Except for a minimal retardation in the body weight gain of the dams during the latter half of the dosing period and a slightly lower overall body weight gain corrected for uterus weight, there was no evidence of an effect of treatment on either maternal or fetal parameters.
On the basis of these results:

Corrected body weight gain were 5.4% and 3.8% in control and treated group, respectively, indicating maternal toxicity at 1000 mg/kg bw/day.

NOAEL maternal < 1000 mg/kg based on reduced bw gain
NOAEL developmental > 1000 mg/kg bw/day

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982-12-13 to 1984-06-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

long dosing period (6 - 27 days) and limited statistical analysis. There were also some limitations in the reporting of the results. The number of reproduction parameters remained unclear at 150 and 450 ppm.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Light beige-colored fine powder

Species:

rabbit

Strain:

Chinchilla

Remarks:

Chinchilla hybrid

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: between 4 and 6 months
- Weight at study initiation: 2975 - 4175 g
- Housing: During the experiment, the animals were housed individually in an "Ehret-Battery"
- Diet: pelleted standard rabbit maintenance diet, ad libitum.
- Water: ad libitum
- Acclimation period: 2 weeks under test conditions, minimum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2 °C
- Humidity: 55±10 %
- Air changes: 12 times/hour (approx.)
- Photoperiod: 12 hours/day

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was dosed by the oral route (gavage) as a suspension in carboxymethylcellulose at a dose volume of 4 mL/kg bw. The suspension of the test article was prepared daily before administration using a homogenizer. A solution of 2 X CMC* in distilled water was used as the vehicle.

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg bw (constant dose volume)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the treatment period three chemical analyses of the stability and homogeneity of the test article suspensions were performed. Achieved concentrations of test material were acceptable, overall mean values were within 104 - 113.6% of nominal values.

Details on mating procedure:

- Impregnation procedure: Females were mated with males of proven fertility.
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Each female was paired with the same male twice, with a separation time of approximately 1 hour.
- The day of observed mating was considered to be day 0 after mating.

Duration of treatment / exposure:

Rabbits were dosed from Day 6-27 after mating

Frequency of treatment:

Once daily

Duration of test:

On day 28 after mating dams were necropsied and the fetuses removed by caesarean section.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Group1)

Dose / conc.:

50 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

150 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

450 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

16 females/group

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a range-finding study.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were performed twice daily

MORTALITY: Yes
- Observations were performed twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily.
The corrected body weight gain mas calculated to be the difference between weights on the first day of treatment and weights on the day of necropsy. Maternal body weight on the day of necropsy was corrected by subtracting the weight of the gravid uterus from the total body weight. Only dams with live fetuses on day 28 p.c. mere used for the calculations of the average weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- on days 6, 11, 15, 19, 24 and 28 of pregnancy Only dams with live fetuses on day 28 p.c. mere used for the calculations of food consumption.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #28 after mating. Dams were killed by cervical dislocation and any fetuses were removed by caesarean section.
- Organs examined: Following examinations of organs, ovaries (number of corpora lutea), uterus (mucosa, contents, amniotic fluid, placentae, abortions, resorptions), the fetuses were identified by uterine position and removed, weighed and inspected externally. The situs of the body cavities (thorax, abdomen pelvis), sex and craniums (after removing the skin) were examined individually from fetuses and investigations of cephalic viscera and brain were performed by the technique of Wilson. The skeletons of the fetuses, including limbs were investigated using Alizarin red (technique of Dawson).

Ovaries and uterine content:

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

Fetal examinations:

The mean fetal body weights, Skeletal investigations

Statistics:

Mean values and standard deviations were applied, whenever feasible. For the sex ratio of fetuses, Chi-Square test was applied. In the Addendum to the Report, statistical significance of skeletal examination was analysed by Fisher’s exact test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No sign of toxicity or clinical symptom urns evident in any
female of group 1 or 2.
In group 3, one female aborted all fetuses on day 27 p.c. (post coitum): five dead fetuses and two live fetuses were found. On day 28 p.c. (day of caesarean section), eight empty implantation
sites were evident in the uterus. In group 4, one female aborted between days 27 and 28 p.c. (four fetal resorptions were found).
On day 28 p.c. (day of caesarean section), nine empty implantation sites and one implantation site with a placenta were evident. In addition, one female aborted between days 27 and 28 p.c. (two implantations were noted, but the stage could not be judged). On day 28 p.c. (day of caesarean section), five live fetuses, one placenta and one empty implantation site were evident. No other sign or symptom which was considered to be related to the treatment with the test article was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight gain from day 6 to day 28 post coitum was reduced by 2.1%, 2.2% and 8.0% in females at 50, 150 and 450 mg/kg bw/day, respectively, when compared to the control group. The mean corrected weight gain in percent of the weight on day 6 were -2.6, -3.1 -4.3 -7.1 at 0, 50, 150 and 450 mg/kg bw/day, respectively. The decrease in corrected bw gain corresponds to 19 %, 65 % and 170 % compared with control animals at 50, 150 and 450 mg/kg bw/day, respectively, when calculated based on % corrected change given in the table below. When calculated based on the change in g, the dases in corrected bw gain are 12 %, 60% and 160% compared with control. In females at 50 mg/kg bw/day, the mean body weight was only reduced on days 2 and 3 (ca 2 %) and in females at 150 mg/kg bw/day from day 2 to day 8 of the treatment ( around 2 %). After these time periods, the body weights increased at 50 and 150 mg/kg bw/day and were comparable with the value of the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of groups 2 and 3 was similar to that of the control group. In group 4, the food consumption mas reduced by 25.67. during the treatment period when compared to that of group 1. During the first recording period without treatment, the food consumption of all groups was similar. Statistical significance of the results was not reported.

Key result

Dose descriptor:

NOAEL

Effect level:

< 50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal body weights were reduced in a dose related manner by 10.5%, 12.7% and 31.6% at 50, 150 and 450 mg/kg bw/day, respectively. Statistical test results were not reported. However, the values for mean body weight of live fetuses obtained from 50 and 150 mg/kg bw/day dose groups in this study were within the range of historical control values of the investigating laboratory.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no significant difference in the sex ratio of any group

External malformations:

no effects observed

Description (incidence and severity):

No abnormal findings were observed externally and in investigations of body cavities (including the craniums) and in cephalic viscera investigations.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal investigations revealed one control fetus with 2 fused sternebrae, one control fetus with longitudinal split sternebrae, and one fetus at 50 mg/kg bw/day had a branched rib. Unsuspicious retardation of skeletal stage was observed in all treated groups. The number of non-ossified phalangeal nuclei in the fore and/or hindlimbs was statistically significantly increased in fetuses of all treated groups. On a litter basis, significant effects were observed almost exclusively only in the highest dose group.

Minor skeletal malformations included rib alterations (supernumerary ribs) at 150 mg/kg bw/day and statistically not significant increases in abnormal ossified strenebrae. The incidence of fetuses with non-ossified sternebra No. 5 was 19%, 22%, 24% and 39% at 0, 50, 150 and 450 mg/kg bw/day. Overall, the skeletal investigation revealed significant effects almost only at the highest, maternal toxic, dose.

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

See the result's tables attached in "Overall remarks, attachments"

Conclusions:

Maternal toxicity was clearly observed as reduced body weight gain and reduced food consumption at 450 mg/kg bw/day. The NOAEL for maternal toxicity was 150 mg/kg bw/day. Increased post-implantation loss and clearly reduced fetal body weight could be observed at 450 mg/kg bw/day. Unequivocal references to embryotoxic effects were lacking, because the post-implantation loss and the reduced fetal body weight may had been caused by primary maternal toxicity. Based on the available data, the NOAEL for developmental toxicity was 150 mg/kg bw/day. The study was acceptable in spite of some shortcomings in reporting.

Executive summary:

The purpose of this study was to evaluate the embryotoxic, fetotoxic and/or teratogenic potential of DESMEDIPHAM, when administered by oral gavage to mated Chinchilla rabbits (hybrid) at dose levels of:
Group 1: 0 mg/kg body meight/day
Group 2: 50 mg/kg body meight/day
Group 3-. 150 mg/kg body meight/day
Group 4: 450 mg/kg body meight/day
On days 6 through 27 after mating (16 mated female rabbits per group).

 

DATA OF DAMS
MORTALITY RATE:
No death occurred during this study in any female of any group.

SIGNS AND SYMPTOMS:
No sign of toxicity or clinical symptom was evident in any female of group 1 or 2. In group 3, one female aborted all fetuses. In group 4, one female aborted all fetuses and one female partially aborted.

FOOD CONSUMPTION:
The food consumption of the group 4 dams uias reduced during the treatment period by 25.6% when compared to that of group 1.

BODY WEIGHT:
In groups 2 and 3, reduced mean body weights from the second to the third treatment day and from the second to the fourth treatment day were recorded, respectively. Increased mean body weights were noted thereafter, and mere similar to that of group 1. In group 4, the mean body meight from day 2 until day 8 of treatment decreased daily. After this period, the body meight increased, although it mas less than that of group 1.

REPRODUCTION DATA:
The postimplantation loss of the group 4 females was increased when compared to that of group 1. This finding was considered to be caused by the higher number of abortions in the group 4 dams. No other difference in the reproduction data was noted.

DATA OF FETUSES
BODY WEIGHT:
A dose-related reduction of body weights was evident in all treated groups when compared with those of group 1. Body weights were reduced by 10.5% in group 2, 12.7% in group 3 and 31.6%
in group 4.

SEX RATIO:
No significant difference was evident between any group.

MALFORMATIONS AND/OR ANOMALIES BY EXTERNAL INVESTIGATIONS:No abnormal finding was evident in any group.

SITUS INVESTIGATIONS OF THE BODY CAVITIES:
No abnormal finding was evident in any group.

INVESTIGATIONS OF THE CRANIUMS AFTER REMOVING THE SKIN:
No abnormal finding was evident in any group.

SKELETAL INVESTIGATIONS:
In group 1, one fetus with a branched rib and fused sternefarae and one fetus with a longitudinally split sternebra was found.
In group 2, one fetus with a branched rib was found.
No malformation or anomaly was found in group 3 or 4.

CEPHALIC VISCERA INVESTIGATIONS BY THE WILSON TECHNIQUE:
No abnormal finding was evident in any group.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-05-22 to 1991-10-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

white powder stored in the dark at ambient temperature

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: ca 4-6 months
- Weight at study initiation: ca 3.7 kg
- Housing: The animals were housed singly in aluminum grid bottomed cages measuring 48 x 61 x 46 cm. Excreta were collected on a peat covered tray, suspended beneath each cage. A separate stainless steel food hopper was provided for each cage, together with a drinking nozzle connected to an automatic watering system. The cages were suspended in racks, each containing 3 rows of 3 cages.
Excreta collection trays were cleaned twice weekly. Cage grids were changed as required. Accessible external surfaces and cage racks were cleaned once weekly.
- Diet: Standard Rabbit Diet, ad libitum until Day 3 of gestation; thereafter a weighed quantity of 250 g was available to each animal on each day.
- Water: domestic mains water, ad libitum.
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18°C ± 3°C
- Humidity: 55% ± 10%
- Air changes (per hr): 15-20
- Photoperiod: There was automatic control of light cycle, Light hours were 0700-1900 h.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

DIET PREPARATION
Standard Rabbit Diet was available to the animals ad libitum until Day 3 of gestation; thereafter a weighed quantity of 250 g was available to each animal on each day. The food was supplied with batch analyses for nutritive constituents and a range of significant contaminants.

FORMULATION OF DOSING SUSPENSIONS
The test material was formulated as a suspension, the vehicle being carboxymethyl cellulose. Fresh dosing suspensions were prepared daily, each concentration being prepared separately. The test material was weighed into a labelled dose container and the requisite quantity of vehicle was added and mixed using a homogeniser. During dosing procedures, suspensions were maintained using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for concentration, homogeneity and stability of dosing formulations were performed prior to commencement of this study. Results of these analyses indicated that formulations could be prepared to a satisfactory standard of accuracy and that they were stable for 24 hours. On the first and tenth days of treatment during the study, 3 x 1 mL aliquots were taken from each of the 3 concentrations of test material and from the Control suspension. These samples were analysed for concentration and homogeneity. Deviations from nominal concentration did not exceed 4%, indicating a satisfactory standard of accuracy in formulation.

Details on mating procedure:

Day 0 was the day of mating. No further info was provided

Duration of treatment / exposure:

6-18 days inclusive of gestation

Frequency of treatment:

Once daily.

Duration of test:

The animals were killed on Day 29 of gestation and the pregnancies were evaluated.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

90 mg/kg bw/day

Dose / conc.:

270 mg/kg bw/day

No. of animals per sex per dose:

16 females/group

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were agreed with the Sponsor after evaluation of the results of a separate dose range finding study.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All the animals were examined for reaction to treatment on each day. The onset, duration and intensity of any signs were recorded, with particular attention being paid to the period 1- 2 h after dosing.

All the animals were checked for viability at the beginning of each day, and again as late as possible on each day. Any animal showing signs of abortion or in poor condition was killed.
These observations included inspection of excreta trays.

BODY WEIGHT: Yes
- Time schedule for examinations:
The animals were weighed on the day of arrival and then on Days 6, 9, 12, 15, 19, 22, 26 and 29 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- The weight of food consumed by each animal, from the 250 g offered each day, was recorded daily, commencing on Day 4 of gestation (weighed quantity first offered on Day 3).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29: Adult animals were killed by intravenous injection of sodium pentobarbitone, via the marginal ear vein. Foetuses were killed by intraperitoneal injection of sodium pentobarbitone. The single animal that aborted its pregnancy prior to Day 29 of gestation was killed on the day on which the abortion was discovered. This animal and the animal which died prematurely during the study were examined at necropsy with a view to diagnosis of each animal's condition. The examination proceeded, where practicable, as described below for necropsies conducted on Day 29 of gestation.

- Organs examined: The contents of the thoracic and abdominal cavities were inspected. Any lesions were described and, where appropriate, representative samples of abnormal tissue were preserved in neutral buffered 10% formalin. During the course of the necropsies, an apparent enlargement of the spleen was noted for the first High dose group animal. Spleen weights of all animals were therefore recorded. The reproductive tract was removed and weighed intact, then opened and the contents were examined. The number of corpora lutea graviditatis in each ovary and the number and position of all implant sites in the uterus were recorded. Each implant was classified as being live, a foetal death (death judged to have occurred after ca Day 18 of gestation), a late embryonic death (embryonic remains visible), or an early embryonic death (only placental remains visible). The borderline between early and late embryonic deaths in this classification lies at ca Day 12 of gestation.

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:

None

Fetal examinations:

Foetuses (including foetal deaths) were examined for externally visible abnormalities. Each viable foetus was individually identified within the litter and weighed. Prior to fixation, the viable foetuses were examined macroscopically for abnormalities of the palate, and of the eyes and cranial bones following removal of the skin over these regions. Two thirds of the viable foetuses from each uterus were fixed in methylated ethyl alcohol and the remaining third in Bouin's fluid; the foetuses were sexed during subsequent dissection procedures. The foetuses fixed in alcohol were subsequently examined for visceral abnormalities by open dissection and the cranium was sectioned once through the coronal suture to allow inspection of the brain in that region. These foetuses were then eviscerated, and the carcasses cleared in potassium hydroxide and glycerol, and the skeletons stained with Alizarin Red S. Skeletal structures in these foetuses were examined for abnormalities and variants, including state of ossification. The foetuses fixed in Bouin's fluid were examined for soft tissue abnormalities by means of whole-body dissection, the internal head structures being examined by means of a free-hand razor blade sectioning technique.

Statistics:

At the discretion of the Study Director, statistical analyses were performed to determine the statistical significance of observed differences between treatment groups. Spleen weights were analysed by analysis of variance. Treatment means were compared by the F-protected Least Significant Difference procedure. Because there was no clear relationship between Day 29 of gestation body weight and spleen weight, analysis of covariance using this body weight as covariate was not considered appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, 12 out of the 14 pregnant animals had reduced faecal output during the treatment period. This finding was concomitant with periods of reduced food consumption. The incidences of reduced faecal output at 30 and 90 mg/kg bw/d were too small to be attributable to treatment. At 30 mg/kg bw/d, one animal aborted its pregnancy on Day 22 of gestation. This animal had shown a marked reduction in food consumption and a reduction in body weight. Immediately prior to necropsy on Day 29 of gestation, one animal in this group, aborted a foetus. The reason for this was not established, but it was not considered to have been related to treatment. Other occasional clinical observations and necropsy findings were considered to have been incidental.

Description (incidence):

There was no mortality

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, there was a reduction in mean body weight gain over the treatment period compared to Control, with a decrease in mean body weight over Days 6 to 12 of gestation.
Body weight performance at 90 and 30 mg/kg bw/d, was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, mean food consumption was 49% lower than Control over the treatment period. The effect was most pronounced over Days 6 to 15 of gestation. Food consumption at 90 and 30 mg/kg bw/d was essentially similar to the Control value.

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:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, an apparent increase in spleen size was observed in the first High dose animal. Spleen weights were therefore recorded for all animals. There was a slight increase in group mean spleen weight at 270 and 90 mg/kg bw/d. These increases did not achieve statistical significance and were considered to be of equivocal biological significance. The lower mean body weight at 270 mg/kg bw/d was not considered to have influenced the interpretation of the spleen weight data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

An animal at 30 mg/kg bw/day dose level was killed prematurely. During the dosing procedure, red liquid was found on the catheter and the animal had dyspnoea. At necropsy, dark red lung lobes were seen. The findings were consistent with accidental damage during the dosing procedure.

Other occasional clinical observations and necropsy findings were considered to have been incidental.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day, one animal aborted its pregnancy on Day 22 of gestation. This animal had shown a marked reduction in food consumption and a reduction in body weight. Histopathological examination revealed findings in the lung consistent with a mild reaction to an inhaled foreign body, and liver changes consistent with a period of inappetence. The aborted pregnancy was considered to have been incidental and not related to treatment with Desmedipham.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

At 30 mg/kg bw/d, one animal aborted its pregnancy on Day 22 of gestation. This animal had shown a marked reduction in food consumption and a reduction in body weight. Histopathological examination revealed findings in the lung consistent with a mild reaction to an inhaled foreign body, and liver changes consistent with a period of inappetence. The aborted pregnancy was considered to have been incidental and not related to treatment with Desmedipham.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Group values for live and dead implants, after exclusion of the above animal, were within the normal background range reported in this laboratory.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, one animal resorbed its litter during early gestation. This animal had a low body weight and slightly low food consumption, prior to commencement of treatment. Resorption of the litter was considered to have been incidental and not related to treatment with Desmedipham.

Dead fetuses:

no effects observed

Details on maternal toxic effects:

Maternal toxicity was demonstrated at 270 mg /kg bw/d by body weight loss and markedly reduced food consumption, and by reduced faecal output. At both 270 and 90 mg kg bw/d there was a slight increase in mean spleen weight. The biological importance of this finding was uncertain.
At 90 mg/kg bw/d, maternal effects were confined to a slightly increased mean spleen weight.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Maternal NOAEL based on rediced spleen weight at 90 mg/kg bw/d; reduced weight gain, reduced food consumption and clinical signs at 270 mg/kg bw/d

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, there was some evidence of slightly retarded foetal development indicated by slightly reduced mean foetal weight, with the lowest weight litter demonstrating retardation of skeletal ossification. This effect was probably related to treatment and may have been secondary to the maternal toxicity.

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):

The incidences of the various abnormalities and variants did not indicate an adverse effect of treatment with Desmedipham.

Skeletal malformations:

no effects observed

Description (incidence and severity):

At 270 mg/kg bw/d, group incidences of the various skeletal ossification parameters indicated slight retardation of skeletal ossification.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

At 270 mg/kg bw/d, group incidences of the various skeletal ossification parameters indicated slight retardation of skeletal ossification. This effect however was essentially due to one litter, which showed marked retardation of skeletal ossification and reduced foetal weight. When this litter was excluded from consideration, the group incidences at this level were essentially similar to the control values. The incidences of the ossification parameters at 90 and 30 mg/kg bw/d did not indicate any effect of treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Remarks on result:

other: Fetal NOAEL based on increased early embryonic death

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

See "Overall remarks, attachments" for the result's tables

Conclusions:

There was no evidence of teratogenicity in this study. Maternal toxicity was clearly observed at the dose level of 270 mg/kg bw/d as a significant reduction in mean body weight gain and markedly reduced food consumption and reduced fecal excretion. The mean spleen weights were increased at 90 and 270 mg/kg bw/d, indicating hemolytical effects of desmedipham. Therefore, as a conservative approach, the maternal NOAEL can be settled at 30 mg/kg bw/d. Desmedipham was embryotoxic at 90 and 270 mg/kg bw/d. The observed effects included increased percentages of early embryonic deaths. Fetotoxicity was manifested as reduced fetal body weight at 270 mg/kg bw/d. The fetuses were considered not to be affected at treatment levels of 30 or 90 mg/kg bw/day. Due to the embryotoxic effect of desmedipham at 90 mg/kg bw/d, the NOAEL for developmental toxicity was 30 mg/kg bw/d.

Executive summary:

Mated female New Zealand White rabbits were randomised into 4 treatment groups, each containing 16 animals. These animals were dosed by gavage once daily over Days 6-18 inclusive of gestation, where Day 0 was the day of mating. Dose levels of test material were as follows:

Group	mg Desmedipham/kg bw/day
Control	0
Low dose	30
Intermediate dose	90
High dose	270

The test material was administered as a suspension, the vehicle being carboxymethyl cellulose.
The animals were monitored during gestation for clinical signs of toxicity, including body weight performance and food consumption.

The animals were killed on Day 29 of gestation and the pregnancies were evaluated. The foetuses were examined for visceral and skeletal abnormalities and variants.

Maternal toxicity was demonstrated at 270 mg Desmedipham/kg bw/day by body weight loss and markedly reduced food consumption, and by reduced faecal output.

At both 270 and 90 mg Desmedipham/kg bw/day, there was a slight increase in mean spleen weight. The biological importance of this finding was uncertain.

In conclusion, the developmental NOAEL is 30 mg/kg bw/d based on slight caudal pelvic shift.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-09-07 to 1985-12-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

The study was conducted according to OECD Guideline 414 and Directive 87/302 EEC Part B guidelines with some exceptions. Statistically significant results were mainly described in the statistical report but the significance was not marked in the summary tables. Additionally, at the end of the dosing period blood samples were collected and Heinz bodies and methemoglobin were determined.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species Wistar Rat, KFM-HAN ., outbred, SPF-Quality.
Rationale Recognized by the international guidelines as the recommended test system.
Number of animals 140 mated females, 35 per group
Initial age (at pairing) Between 11 and 12 weeks
Initial body weight (post coitum) 190 - 270 g
Acclimation 9 days (minimum) under test conditions, after veterinary examination .
Identification Individual cage number and corresponding ear tag .

HUSBANDRY
Conditions The animals were housed under Standard Laboratory Conditions: air-conditioned with 10-15 air changes per hour, and hourly monitored environment with temperature 22 +/- 2 degrees centigrade, relative humidity 55 +/- 10 %1 12 hours artificial fluorescent light/12 hours dark, and at least 8 hours music/light period. Neither insecticides nor chemicals were applied in the animal rooms 1 with the exception of a disinfectant for floor and wall cleaning once weekly.
Accommodation
Individually in Makrolon type-3 cages with standard softwood bedding (lignocel, Schill AG/ Switzerland) Throughout the study 1 each cage was identified by a colored label, which recorded the group , the study schedu1e number, anima1 number and details of treatment.
Diet
Pelleted standard Kliba 343 rat maintenance diet was provided ad libitum
Water
Tap water was available ad libitum.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Distilled water with 2 % CMC

Details on exposure:

The test article was administered orally by intubation, once daily in the morning from day 6 through day 15 post coitum. All groups received a volume of 10 ml/kg body weight with a daily adjustment of individual volume to the actual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of concentration. and homogeneity of the applied test article/vehicle mixtures of all dose groups were performed twice during the treatment period (at initiation and termination).

Details on mating procedure:

MATING
After acclimation, the females were housed with males (1:1) overnight until either the daily vaginal smear was sperm-positive or a copulation plug was observed. After this time, the female was removed and rehoused individually in a separate cage. The day was recorded as day O post coitum.

Duration of treatment / exposure:

From day 6 to day 15 post coitum.

Frequency of treatment:

Once daily during exposure period.

Duration of test:

21 days post coitum, ending with CO2 asphyxiation.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

35

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages were based upon the results of a preceding embryotoxicity (including teratogenicity) study (RCC Project 024996, reported on June 6, 1985) in which two anomalied fetuses in group 3 (100 mg/kg) and eight anomalied fetuses in group 4 (1000 mg/kg) were found. Therefore, to determine if the findings in group 3 (100 mg/kg) were incidental, this dosage was used in this study as well. In the high dose group of the preceding embryotoxicity study, it could be possible that the dosage of 1000 mg/kg which caused distinct maternal toxicity may have resulted in the anomalies noted in that study. For clarification, in this study 1 the top dose was decreased to 500 mg/kg.
- Fasting period before blood sampling for (rat) dam thyroid hormones: Not specified.
- Time of day for (rat) dam blood sampling: On day 16 post coitum, blood samples were collected from all animals between the hours 7.30 and 9.00 a.m. to reduce biologic variation caused by circadian rhythms.

Maternal examinations:

MORTALITY
Twice daily, minimum.
SIGNS AND SYMPTOMS
Twice daily, minimum.
BODY WEIGHTS
Daily, from day O until day 21 post coitum.
FOOD CONSUMPTION
The data were recorded on day 6, 11, 16 and 21 post coitum.

Ovaries and uterine content:

CAESAREAN SECTION AND POSTMORTEM EXAMINATIONS
The investigations of dams and fetuses were performed in accordance with international recommendations (e g. US-EPA guidelines). Following examination of the organs, ovaries (number of corpora utea), uterus and uterus contents, the fetuses were identified by uterine position and removed, sexed, weighed, examined externally and submitted to one of the following procedures individually:

The uteri (and contents) of all pregnant females were weighed on the scheduled day of necropsy and used to determine the corrected body weight gain (see Data Compilation and Data Processing on the next page).
The uteri of all females which were found at necropsy to be not pregnant were placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.

Blood sampling:

On day 16 post coitum, blood samples were collected from all animals between the hours 7.30 and 9.00 a.m. to reduce biologic variation caused by circadian rhythms. Blood samples were collected from the retro-orbital plexus, except females nos. 1-4 of the vehicle control group, in which blood samples were collected from the tail vein. The reason for changing the method is described on p. 19 under signs and symptoms.

Heinz bodies were examined using microscopy.
Methemglobin was examined using a IL 282 Co-Oximeter.

Fetal examinations:

One half of the total number of fetuses were fixed in a solution of ethyl alcohol, formaldehyde and acetic acid. The selected fetuses were cross-sectioned and the viscera were examined*. A description of any abnormality was recorded. After examination, each fetus was preserved in a solution of ethyl alcohol and glycerine in the ratio 1:3; one fetus/bottle.
The remaining half of the total number of fetuses were placed in a solution of potassium hydroxide for clearing and stained with alizarin red (modified technique). The skeletal structure of each fetus was examined and a description of any abnormality was recorded After examination, the individual fetuses were preserved in plastic bags.

Statistics:

DATA COMPILATION
Body weight, food consumption and caesarean section data were recorded on-line and evaluated by computer. All other data were recorded on data sheets and evaluated manually.

DATA PROCESSING
Corrected body weight gain was calculated using the formula.
Body weight on gestation day 21 - Body weight on gestation day 6 - Uterus weight at necropsy on gestation day 21 = Corrected body weight gain.

The mean daily food consumption was calculated using the
following formula:
Grams of food consumed per period / Days per period = Mean daily food consumption (g)

Only dams with live fetuses on day 21 post coitum were included in the calculations of body weight gain, corrected body weight gain, mean daily food consumption and reproduction data.
The computer-generated values which appear in the tables represent the rounded-off results of calculations which used the exact raw data values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

SIGNS AND SYMPTOMS:

No test article-related signs or symptoms were observed in any female of any dose group.

Group 1 (0 mg/kg):
In female no. 4, colporrhagia was noted during blood collection from the tail vein on day 16 post coitum, caused by panic attempts to escape from the immobilization box. (This was the reason to collect the blood in all following animals from the retro-orbital plexus.) In female no. 16, the right eye was injured during blood collection from the retro-orbital plexus.

Group 2 (10 mg/kg):
In female no. 42, loss of body weight was noted from day 13 to day 14 post coitum caused by prevention of water consumption during the night, due to water bottle leakage.

Group 3 (100 mg/kg):
No sign or symptom was observed in any female of this group.

Group 4 (500 mg/kg):
In female no. 121 1 ruffled fur was noted on day 11 post coitum. In female no. 126, the right eye was injured during blood collection from the retro-orbital plexus. In females nos. 130 and 131, hairless regions on the abdomen or on the left side of the body were noted on day 17 and 16, post coitum, respectively.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHTS:
The mean body weight gain and the mean corrected body weight gain of groups 2 (10 mg/kg) and 3 (100 mg/kg) were similar to those of Group 1 (0 mg/kg). In group 4 (500 mg/kg) 1 significantly reduced mean body weight gain was noted during the treatment period, when compared to that o f group 1 ( 0 mg/ kg) . During this period I the mean body weight gain of group 1 was 20.1 %, group 4 amounted to 8.4 %. Significant differences between these groups were also noted in the mean corrected body weight gain. The values of group 1 and 4 were 5.1 %. and -1.5 %, respectively. These findings were considered test article- related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION:
The mean food consumption during the different recording periods was similar in groups 1 (0 mg/kg) 1 2 (10 mg/kg) and 3 (100 mg/kg). In group 4 (500 mg/kg), the mean food consumption was significantly reduced (21.3 %) during the treatment period, when compared to that of group 1 (0 mg/kg). This finding was considered test article related.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

CLINICAL LABORATORY INVESTIGATIONS:
Hematological data indicated toxic methemoglobinemia at day 16 post coitum. This was characterized by a significant increase in Heinz bodies in the rats of group 4 (500 mg/kg) and methemoglobin formation in the rats of groups 3 (100 mg/kg) ·and 4. Although not statistically significant, a trend for increased methemoglobin formation was also noted in the rats of group 2 (10 mg/kg).

Gross pathological findings:

no effects observed

Description (incidence and severity):

NECROPSY FINDINGS,
No abnormal finding in any female of any group was evident.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA:
No test article- or dose-related differences in the mean number of implantations, fetuses or embryonic deaths were evident between groups. The mean distribution of fetuses and resorptions within the uterus horns were similar in all groups.

Details on maternal toxic effects:

During the course of this study, no deaths occurred and no treatment related signs or symptoms were observed. Maternal toxicity was indicated by significantly reduced body weight and corrected body weight gain and food consumption at 500 mg/kg bw/day during the treatment period. The mean body weight gains were 20.1% and 8.4% at 0 and 500 mg/kg bw/day, respectively (see Table: Mean differences in body weight gain of dams). The corrected body weight gains were 5.1%, 5.4%, 4.7% and -1.5% at 0, 10, 100 and 500 mg/kg bw/day, respectively. At 500 mg/kg bw/day, the mean food consumption was significantly reduced ( 21.3%) compared to that of the control group. Hematological data indicated methemoglobinemia. There was a significant increase in methemoglobin formation on day 16 of gestation in dams at 100 (p<0.01) and 500 mg/kg bw/day (p<0.01) and a significant increase in Heinz bodies in dams at 500 mg/kg bw/day (p<0.01). A trend for increased methemoglobin formation was noted in treated dams, the values were 1.3%, 1.6%, 3.7% and 9.3% at 0, 10, 100 and 500 mg/kg bw/day, respectively (Table: Hematology - females).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

haematology

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHTS:
The mean body weight of groups 1, 2 and 3 was similar. The group mean body weight of the group 4 (500 mg/kg) fetuses was significantly reduced (by 10.6%) when compared to that of group 1 ( 0 mg/ kg) . This finding was considered test article-related .

Changes in sex ratio:

no effects observed

Description (incidence and severity):

SEX RATIOS:
No significant differences in the sex ratio of group 2 (10 mg/kg), 3 (100 mg/kg) or 4 (500 mg/kg) were evident when compared to that of group 1 ( 0 mg/ k g ) .

External malformations:

effects observed, treatment-related

Description (incidence and severity):

MALFORMATIONS AND/OR ANOMALIES BY EXTERNAL INVESTIGATIONS (p. 28):
Group 1 (0 mg/kg):
No finding in any fetus was evident.
Group 2 ( 10 mg/ k g ) :
No findings in any fetus was evident.
Group 3 ( 100 mg/ k g ) :
One fetus (no. 629) of dam no. 83: Runt with 2.2 g body weight (0.2%) .
One fetus (no. 1210) of dam no. 98: Hydrops (generalized) <0.2 %).
Total number- of fetuses affected: 2 (0.4 %).
Group 4 (500 mg/kg):
One fetus (no. 1322) of dam no. 131: Micrognathia (inferior) (0.3%) .

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

In skeletal investigations of fetuses malformations and abnormalities included absent sternebra (nos. 5, 6), abnormally shaped sternebrae (nos. 4, 5) and wavy ribs. These skeletal anomalies were seen in all groups (Table: Summary of reproduction, malformations and anomalies in Wistar rats). In the comparison of the stage of skeletal development, there was an increase in incompletely or still absent ossifications of sternebrae, phalangeal nuclei, calcanea and cervical vertebrae in fetuses at 500 mg/kg bw/day as compared with the control group (Table: Skeletal investigation of fetuses).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

VISCERAL INVESTIGATIONS OF FETUSES BY THE WILSON TECHNIQUE:
No test article-related finding was noted in any fetus of any dose group. The isolated findings noted were common and incidental in rats of this rat strain used.
In group 1 (0 mg/kg) and 2 (10 mg/kg) 1 one fetus with coagulated blood within the stomach was noted, respectively.
In group 3 (100 mg/kg), one fetus with dilated renal pelvis (right kidney) was ascertained.
In group 4 (500 mg/kg) 1 one fetus with coagulated blood within the stomach, and one fetus with coagulated blood within the occiput was noted.

Details on embryotoxic / teratogenic effects:

The mean fetal body weight was significantly reduced (10.6 %) at 500 mg/kg bw/day. At 100 mg/kg bw/day, two fetuses with major malformations (runt, hydrops) were observed and one fetus had micrognathia inferior at 500 mg/kg bw/day. In the visceral investigations one fetus with renal pelvis dilated was observed at 100 mg/kg bw/day. One fetus with coagulated blood within the stomach was observed at 0, 10 and 500 mg/kg bw/day and one fetus with coagulated blood within the occiput at 500 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

70 mg/kg bw/day

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:

not specified

Relevant for humans:

not specified

NUMBER OF DAMS PER GROUP USED FOR CALCULATIONS:
Group 1 ( 0 mg/kg) : 33 dams
Group 2 ( 10 mg/kg) : 32 dams
Group 3 ( 100 mg/kg) : 35 dams
Group 4 ( 500 mg/kg) : 32 dams

Only dams with living fetuses (one fetus minimum) on day 21 post coitum were used for the calculations of body weight gain, food consumption and reproduction data.

NUMBER AND REASONS FOR EXCLUSION OF FEMALES FROM EVALUATIONS:
Group 1 < 0 mg/kg):
Two females (nos. 19 and 31) were not pregnant.
Group 2 ( 10 mg/kg):
Three females· (nos. 49, 53 and 66) wer-e not pregnant.
Group 3 ( 100 mg/kg):
All females were pregnant and used for calculations.
Group 4 ( 500mg/kg):
Three females (nos. 117, 118 and 121) were not pregnant.

 

Conclusions:

Because of the low achieved concentrations down to 70 % of nominal concentration, doses were corrected to 7, 70 and 350 mg/kg bw /day.
Maternal NOAEL : 7 mg/kg bw/day based on increased level of methemoglobin at 70 mg/kg bw/day.
Developmental NOAEL : 70 mg/kg bw/day based on reduced fetal body weight and reduced state of skeletal ossification shown as an increase in incompletely ossified sternebrae and absent ossification of phalangeal nuclei, calcanea and cervical vertebrae.

Executive summary:

The purpose of this embryotoxicity (including teratogenicity) study was to assess the effects of DESMEDIPHAM TECHNICAL upon embryonic and fetal development when administered orally by intubation once daily to mated female Wistar rats (outbred, SPF-quality) from day 6 through day 15 post coitum at dose levels of 0, 7, 70 and 350.
Each group consisted of 35 mated female rats. The dosages were based upon the results of a preceding embryotoxicity (including teratogenicity) study in the rat <RCC Project 024996, reported on June 6, 1985).
On day 16 post coitum blood samples were collected from the retro- orbital plexus and investigated for Heinz bodies and methemoglobin. The hematological data indicated toxic methemoglobinemia. This was characterized by a significant increase in Heinz bodies in the rats of group 4 (350 mg/kg) and methemoglobin formation in the rats of groups 3 (70 mg/kg) and 4. Although not statistically significant, a trend for increased methemoglobin formation was also noted in the rats of group 2 (7 mg/kg).
On day 21 post coitum, all females were sacrificed and the fetuses removed by caesarean section. The investigations of females/dams and fetuses were performed in accordance with international recommendations. All parameters recorded were evaluated and reported. From this study, the following results were obtained:
No death occurred and no test article-related signs or symptoms were observed. At necropsy, no findings were evident in any female of any group.
The oral administration of DESMEDIPHAM TECHNICAL caused significantly reduced body weight gain and food consumption in the dams of group 4 (350 mg/kg) during the treatment period. Additionally, the corrected body weight gain was significantly lower in this group.
No test article- or dose-related differences between the vehicle control group and any dose group were noted in any of the reproduction data recorded.
The evaluation of the data of fetuses yielded significantly reduced mean body weights in group 4 (350 mg/kg) in comparison with that of the control group.
During the external investigations of fetuses in group 3 (70 mg/kg), two anomalied fetuses were found. One fetus of dam no. 83 was a runt and one fetus of dam no. 98 was a hydrops. In group 4 (350 mg/kg), one fetus had micrognathia inferior. The type and frequency of these findings were considered to be incidental and common in rats of this strain and age.
No test article-related findings were ascertained during the visceral investigations of fetuses by the Wilson technique. The isolated findings of coagulated blood within the stomach (one fetus in groups 1, 2 and 4, respectively, dilated renal pelvis of the right kidney (one fetus of group 3) and coagulated blood within the occiput (one fetus of group 4) were common and incidental of nature.
The skeletal investigations of fetuses for anomalies resulted in similar findings of nonspecific variations in all groups. The comparison of the stage of skeletal development yields an increased quota of incompletely or still absent ossifications of sternebrae, phalangeal nuclei, calcanea and cervical vertebrae in the fetuses of group 4 (350 mg/kg). This result corresponded to the reduced body weights of fetuses.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-09-07 to 1985-12-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

According to the study report, this study was conducted in compliance with GLP regulations, with the possible exceptions of minor items, none of which could be considered to effect the validity of the data or interpretation of the results in the report. These exceptions were, however, not clearly identified in the study report.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

The study was conducted according to OECD Guideline 414 and Directive 87/302 EEC Part B guidelines. Statistically significant results (except sex ratio of live fetuses) were not reported in summary tables.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species Wistar Rat, KFM-HAN ., outbred, SPF-Quality.
Rationale Recognized by the international guidelines as the recommended test system.
Number of animals 100 mated females, 25 per group
Initial age (at pairing) Between 11 and 12 weeks
Initial body weight (post coitum) 190 - 227 g
Acclimation 9 days (minimum) under test conditions, after veterinary examination .
Identification Individual cage number and corresponding ear tag .

HUSBANDRY
Conditions The animals were housed under Standard Laboratory Conditions: air-conditioned with 10-15 air changes per hour, and hourly monitored environment with temperature 22 +/- 2 degrees centigrade, relative humidity 55 +/- 10 %1 12 hours artificial fluorescent light/12 hours dark, and at least 8 hours music/light period. Neither insecticides nor chemicals were applied in the animal rooms 1 with the exception of a disinfectant for floor and wall cleaning once weekly.
Accommodation
Individually in Makrolon type-3 cages with standard softwood bedding (lignocel, Schill AG/ Switzerland) Throughout the study 1 each cage was identified by a colored label, which recorded the group , the study schedu1e number, anima1 number and details of treatment.
Diet
Pelleted standard Kliba 343 rat maintenance diet was provided ad libitum
Water
Tap water was available ad libitum.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Distilled mater with 2 X CMC * was used as vehicle of the test article in the dose groups and was applied as the control article to the females of the control group.

Details on exposure:

The test article was administered orally by intubation, once daily in the morning from day 6 through day 15 post coitum. All groups received a volume of 10 ml/kg body weight with a daily adjustment of individual volume to the actual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of concentration, and homogeneity of the applied test article/vehicle mixtures of all dose groups were performed twice during the treatment period (at initiation and termination).

Details on mating procedure:

MATING
After acclimation, the females were housed with males overnight until either the daily vaginal smear was sperm-positive or a copulation plug was observed. After this time, the female was removed and rehoused individually in a separate cage. The day was recorded as day 0 post coitum.

See allocation table.

Duration of treatment / exposure:

9 days

Frequency of treatment:

The test article was administered orally by intubation, once daily in the morning from day 6 through day 15 post coitum.

Duration of test:

47 days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dosages were based upon the results of the dose-finding embryotoxicity (including teratogenicity) study (RCC Project 033142, reported on August 10, 1984) in which 1000 mg/kg/day caused slight maternal toxicity indicated by reductions of body weight gain and food consumption in the dams and slightly reduced mean body weights of fetuses. Therefore, 1000 mg/kg/day was selected as the highest dose level and 10 mg/kg/day was considered unlikely to cause toxic symptoms.
- Fasting period before blood sampling for (rat) dam thyroid hormones: Not specified.
- Time of day for (rat) dam blood sampling: 07:30 - 09:00 AM.

Maternal examinations:

MORTALITY
Twice daily, minimum.

SIGNS AND SYMPTOMS
Twice daily, minimum.

DATA RECORDING
The following data were recorded.

BODY WEIGHTS
Daily, from day O until day 21 post coitum (see Data Compilation and Data Processing on the next page).

FOOD CONSUMPTION
The data were recorded on day 6 1 11, 16 and 21 post coitum (see Data Compilation and Data Processing on the next page).

Ovaries and uterine content:

CAESAREAN SECTION AND POSTMORTEM EXAMINATIONS
The investigations of dams and fetuses were performed in accordance with international recommendations (e g. US-EPA guidelines). Following examination of the organs, ovaries (number of corpora utea), uterus and uterus contents, the fetuses were identified by uterine position and removed, sexed, weighed, examined externally and submitted to one of the following procedures individually:

The uteri (and contents) of all pregnant females were weighed on the scheduled day of necropsy and used to determine the corrected body weight gain (see Data Compilation and Data Processing on the next page).
The uteri of all females which were found at necropsy to be not pregnant were placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.

Fetal examinations:

One half of the total number of fetuses were fixed in a solution of ethyl alcohol, formaldehyde and acetic acid. The selected fetuses were cross-sectioned and the viscera were examined*. A description of any abnormality was recorded. After examination, each fetus was preserved in a solution of ethyl alcohol and glycerine in the ratio 1:3; one fetus/bottle.
The remaining half of the total number of fetuses were placed in a solution of potassium hydroxide for clearing and stained with alizarin red (modified technique). The skeletal structure of each fetus was examined and a description of any abnormality was recorded After examination, the individual fetuses were preserved in plastic bags.

Statistics:

DATA COMPILATION
Body weight, food consumption and caesarean section data were recorded on-line and evaluated by computer. All other data were recorded on data sheets and evaluated manually.

DATA PROCESSING
Corrected body weight gain was calculated using the formula.
Body weight on gestation day 21 - Body weight on gestation day 6 - Uterus weight at necropsy on gestation day 21 = Corrected body weight gain.

The mean daily food consumption was calculated using the
following formula:
Grams of food consumed per period / Days per period = Mean daily food consumption (g)

Only dams with live fetuses on day 21 post coitum were included in the calculations of body weight gain, corrected body weight gain, mean daily food consumption and reproduction data.
The computer-generated values which appear in the tables represent the rounded-off results of calculations which used the exact raw data values.

Clinical signs:

no effects observed

Description (incidence and severity):

No test article-related signs or symptoms were observed in any female of any dose group (see necropsy findings also).
Group 1 In female no. 19, hairless regions were noted on day 9 post coitum.
Group 2 ( 10 mg/kg) :
In female no. 47, colporrhagia was observed on day 15 post coitum. This symptom1 in connection with body weight loss 1 suggested abortion This finding was considered to be incidental and not test articlerelated, because no abortion was noted in the 100 mg/kg dose group or in the 1000 mg/kg dose group with distinct maternal toxic effects.
Group 3 (100 mg/kg) :
No sign or symptom was observed in any female of this group.
Group 4 ( 1000 mg/kg):
In female no. 91 1 high body weight loss from day 9 post coitum (231 g) to day 16 post coitum (174 g) was noted. After the treatment period until necropsy on day 21 post coitum1 an above-average body weight gain of 60 g was recorded. These findings were attributed to an administration error and not by the test article.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHTS (pp 39-52>:
The mean body weight gain and the mean corrected body weight gain of groups 2 (10 mg/kg) and 3 (100 mg/kg) were similar to those of group 1 ( 0 mg / k g ) .
In group 4 (1000 mg/kg), significantly reduced mean body weight gain was noted during the treatment period, when compared to that of group 1 ( contro1 ) . During this period , the mean body weight gain of group 1 was 18.9 %; group 4 amounted to 9.7 %. Significant differences between these groups were also noted in the mean corrected body weight gain. The values of group 1 and 4 were 8.1 % and 3.8 %, respectively. These findings were considered test article-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION (pp. 53-S8):
The mean food consumption during the different recording periods was similar in groups 1 (0 mg/kg) 1 2 (10 mg/k g) and 3 (100 mg/kg) .
In group 4 (1000 mg/kg) 1 the mean food consumption was significantly reduced (17.4 %) during the treatment period, when compared to that of group 1 (control) This finding was considered test article related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

NECROPSY FINDINGS
No test article-related findings were evident in any animal of any dose group. The single findings observed in groups 2 and 4 were considered to be incidental and not caused by the test article.
Group 1 ( 0 mg/ kg): No abnormal finding in any female was evident.
Group 2 (10 mg/kg) : In female no. 47, the uterus contained 2 implantation sites only and the corpora lutea were already receded (see also signs and symptoms). No abnormal findings on the other organs were evident.
Group 3 ( 100 mg/kg): No abnormal finding in any female was evident.
Group 4 < 1000 mg/kg): Female no. 91 had 15 corpora lutea, 9 embryonic resorptions and 5 under weight fetuses between 1.90 g and 2.32 g. The following abnormal findings were recorded: whitish-greyish bilateral infiltration of the vertebral column from the cranial region of the thorax to the diaphragm. These findings were attributed to be caused by an administration error with distribution of small amount of the test article/vehicle mixture in the surrounding tissue of the esophagus (see also signs and symptoms)

Dead fetuses:

no effects observed

Description (incidence and severity):

REPRODUCTION DATA (pp. 26-27, 59-62 1 68-98) ·
No test article- or dose-related differences in the mean number of implantations, fetuses or embryonic deaths were evident between any group. The mean distribution of fetuses and resorptions within the uterus horns were similar in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Maternal NOAEL based on bodyweight effects

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight of groups 1, 2 and 3 was similar. The group mean body weight of the group 4 (1000 mg/kg) fetuses was significantly reduced (by 12.5 %) when compared to that of group 1 (0 mg/kg). This finding was considered test article-related.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test article-related differences between the group 1 (0 mg/kg) and the dose groups were evident. The significant difference between the sex ratio of group 1 (0 mg/kg) and group 2 (10 mg/kg) was considered to be incidental and caused by the contrary ratio of male and female fetuses in these groups. No significant differences in the sex ratio of groups 3 (100 mg/kg) and 4 (1000 mg/kg) were ascertained.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

MALFORMATIONS AND/OR ANOMALIES BY EXTERNAL INVESTIGATIONS (p. 31):
Group 1 ( 0 mg/ kg):
No finding in any fetus was evident.
Group 2 ( 10 mg/Kg):
No finding in any fetus was evident.
Group 3 ( 100 mg/kg):
One fetus (no. 207) of dam no. 56: Omphaloce1e (0.4%)
One fetus (no. 309) of darn no. 61: Agnathia (inferior) and "open eye" bilaterally (0.4 %)
Total number of fetuses affected: 2 (0.8%)
Group 4 ( 1000 mg/kg):
One fetus (no. 770) of dam no. 90: Runt with 1.7 g body weight (0.4%)
Fetuses of dam no. 100; six fetuses (nos. 1020, 1022, 1023, 1025, 1026, 1027): Palatoschisis and slight micrognathia (inferior) (2.5%).
One fetus (no. 1024): Palatoschisis, distinct micrognathia (inferior') and a dysplastic tail (slight constriction proximal, lateral curvature distal (0.4%.
Total number of fetuses affected: 8 (3.3%)

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

No test article-related finding was noted in the fetuses of groups 2 (10 mg/kg) and 3 (100 mg/kg). The frequency of the nonspecific findings was higher in group 2 and lower in group 3, in comparison to that of group 1 ( 0 mg/ kg ) In group 4 (1000 mg/kg), the types of anomalies were similar when compared to those of group 1 (0 mg/kg) but a higher frequency was noted. This finding was considered to be test article-related and primary a defective maturation caused by the maternal toxicity. The following non specific skeletal anomalies of fetuses were noted.

Group 1 ( 0 mg/kg) : Ten(10) fetuses (6.9 %) with anomalies were noted in the 144 fetuses investigated: longitudinally split sternebrae 5 and 6 (one fetus); longitudinally split sternebra 5 (three fetuses); longitudinally split sternebrae 3-5 (one fetus); dumbbell-shape thoracic vertebral body 10 (one fetus); dumbbell-shape thoracic vertebral body 11 (one fetus); absent sternebrae 5 and 6 (one fetus); absent sternebra 5 (2).
Group 2 ( 10 mg/kg): Twelve(12) fetuses (10.4 %) with anomalies were noted in the 115 fetuses investigated, abnormally shaped (asymmetric) sternebrae 4 and 5 (two fetuses); longitudinally split sternebra 5 (one fetus); absent sternebrae 1 and 6, abnormally shaped (asymmetric) sternebra 3 Cone fetus); absent sternebra 6, abnormally shaped (asymmetric) sternebrae 3, 4 and 5 (one fetus) ; wavy ribs 1 - 13 ( 1eft and right side) , rib 9 ( right side ) additiona11y was shortened ( one fetus); wavy ribs 11 -13, right side (one fetus) ; longitudina11y split sternebrae 1-3 (one fetus); absent sternebra 6 (one fetus); absent sternebra 5 (two fetuses); dumbbell-shape thoracic vertebral body 12 (one fetus)
Group 3 ( 100 mg/ kg), Two(2) fetuses (1.5 %) with anomalies were noted in the 132 fetuses investigated: longitudinally split sternebrae 5 and 6 (one fetus); longitudinally split sternebrae 3-5 Group 4 (1000 mg/kg) Twenty(20) fetuses (16.8 %) with anomalies were noted in 119 fetuses investigated , wavy ribs 10 - 12 (right side) 1ongitudina11y split sternebrae 4 and 5 (one fetus); longitudinally split sternebrae 3-5 (one fetus); longitudinally split sternebrae 4 and 5 (one fetus); absent sternebrae 5 and 6, longitudinally split sternebrae 2-4 (one fetus); longitudinally split sternebra 5 (three fetuses); longitudinally split sternebrae 2-5 (three fetuses); dumbbell-shape thoracic vertebral body 12 (one fetus); longitudinally split sternebrae 3-6 (one fetus); absent sternebrae 5 and 6 (one fetus); absent sternebra 5 (two fetuses); dumbbell-shape thoracic vertebral body 11 (one fetus); dumbbell-shape lumbar vertebral body 1 (one fetus); absent sternebra 3, longitudinally split sternebra 2, longitudinally split and fused sternebrae 4 and 5 Cone fetus); longitudinally split sternebra 2 1 fused sternebrae 4 and 5 (one fetus); fused sternebrae 4 and 5 (one fetus).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

No abnormal finding was evident in any fetus of group 1 (0 mg/kg) and no test article-related abnormalities were noted in the fetuses of any dose group. In group 2 ( 10 mg/ kg) 1 one fetus with dilated rena1 pe1vis (right and left kidney) and in group 3 (100 mg/kg), two fetuses with dilated renal pelvis (right and left kidney) were ascertained. In group 4 (1000 mg/kg), no additional abnormal finding was noted during visceral investigations, in the four fetuses (of seven) with palatoschisis which were allocated to visceral investigations this finding was confirmed.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

SKELETAL INVESTIGATIONS (STAGE OF DEVELOPMENT):
The stages of skeletal ossification in groups 1, 2 and 3 were similar. The differences noted were within the normal range of deviations for animals of this strain and age. In group 4 (1000 mg/kg)i the quota of incompletely ossified crania (as occipitale) and sternebrae as well as phalangeal nuclei, calcanea and vertebrae with still absent ossification were increased, compared to that of group 1 (0 mg/kg). These findings were considered to be the consequence of the distinctly reduced mean body weight of fetuses and not a specific effect of the test article on skeletal development. Further, for the increased quota of fetuses with a supernumerary rib (bilateral), no explanations were present.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

visceral malformations

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

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:

not specified

Relevant for humans:

not specified

Conclusions:

Maternal toxicity was noted as reduced body weight and corrected body weight gain, and reduced food consumption at 1000 mg/kg bw/day. Based on these parameters the maternal toxicity NOAEL was 10 mg/kg bw/day.
At 100 mg/kg bw/day the number of fetuses with major malformations (palatoschisis and micrognathia) and minor skeletal malformations increased. The number of fetuses with supernumerary rib was increased at 100 and 1000 mg/kg bw/day. In fetuses at 1000 mg/kg bw/day the stage of skeletal ossification and fetal body weight was reduced. These effects could be attributed to either maternal toxicity or incidental events (supernumerary rib).
Based on these results and taking a conservative approach, i.e. taking even events of questionable toxicological relevance at 100 mg/kg bw/day into account, a NOAEL for embryotoxicity of 10 mg/kg bw/day can be derived.
The study was acceptable.

Executive summary:

Desmedipham (Purity 97.8%) was administered to four groups of 25 mated female Wistar (KFMHAN, outbred, SPF-quality) rats at dose levels of 0, 10, 100 and 1000 mg/kg bw/day. The dams received desmedipham once daily over gestational days of 6 to 15. The test article was dosed orally by intubation as a suspension of 2% carboxymethylcellulose in distilled water at a dose volume of 10 ml/kg bw. Achieved test material concentrations were satisfactory with overall mean values between 89.7 and 94.1% at all dose levels at the testing times. The test material was stable in the dosing vehicle with overall mean values between 93.2 and 114.5% over the 90 minute post dosing period. Body weight and food consumption were recorded during the study.

On day 21of gestation the dams were necropsied and the fetuses removed by caesarean section. Ovaries (number of corpora lutea), uterus and its contents were examined. The fetuses were identified by uterine position and removed, sexed, weighed and examined externally. Approximately one half of the fetuses from each litter were fixed (in a solution of ethyl alcohol, formaldehyde and acetic acid), cross-sectioned and the viscera were examined by Wilson’s technique. The remaining half of the fetuses were placed in the solution of potassium hydroxide for clearing, stained with Alizarin red and then examined for skeletal abnormalities and variants. Body weight, food consumption and reproduction data (total number of implantation, number of pups) were statistically analysed (Univariate one-way analysis of variance, Student’s t-test, Wilcoxon Ranks test, Kruskall-Wallis test, Chi-square).

Maternal NOAEL: 10 mg/kg bw/day based on reduced body weight and corrected body weight gain, and reduced food consumption at 1000 mg/kg bw/day and decreased corrected bw gain at 100 mg/kg bw/day
Developmental NOAEL: 10 mg/kg bw/day based on increased number of fetuses with supernumerary rib

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

All studies in the rat and rabbit were performed to GLP and appropriate test guidelines. The studies are not fully compliant with the current test guideline (OECD 414) but are considered to be accpetable (K2) for assessment.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Toxicity to reproduction: other studies

Additional information

The available developmental toxicity studies have recently (2019) been assessed by RAC; the outcome of these assessments is summarised below.

Key rat study: M-146758-01-1 (Study 1), 1985

The top dose level of 1000 mg/kg bw/d caused reduced food consumption (by 17%) during the treatment period.  The corrected body weight was only marginally reduced (~4%).  In addition, haematotoxicity is assumed based on the results of other subsequent studies.  The most remarkable developmental finding in this study was the occurrence of cleft palate and micrognathia in 7 foetuses from the same litter (containing 9 foetuses in total) at the top dose of 1000 mg/kg bw/d.  The severity of micrognathia was ‘distinct’ in 1 foetus and ‘slight’ in the
remaining 6.  General toxicity in the dam producing this litter was not markedly higher compared to other dams of this group. One foetus with agnathia was also observed at the mid-dose of 100 mg/kg bw/d.  In addition, increased incidence of split sternebrae was observed at the top dose; however, presence of split sternebrae in the control group reduces the concern about this anomaly (13 foetuses in 10 litters at the top dose vs 5 foetuses in 5 litters in the control). Likewise, the observed reduction in foetal body weight (by 12%) and delayed ossification in the presence of some maternal toxicity were not considered by RAC to be sufficiently adverse to warrant classification.

Key rat study: M-146758-01-1 (Study 2), 1985

This study was conducted as a follow-up to the previous study to further investigate the mandibular malformations.  Animals of the same strain and source were used.  A lower top dose level was chosen (500 mg/kg bw/d instead of 1000 mg/kg bw/d) to limit the potential confounding effect of maternal toxicity and the group size was increased (35 instead of 25 females).  In addition, MetHb and Heinz bodies were determined as indicators of haematotoxicity.  The top dose level of 500 mg/kg bw/d caused reduced food consumption (by 21%) during the treatment period.  The corrected body weight was reduced by 5%.  A significant increase in MetHb (9%) and Heinz bodies (37%) was observed at the top dose and a small increase in MetHb (4%) was also present at 100 mg/kg bw/d.  No significant developmental findings were observed apart from reduced foetal body weight (by 11%), delayed ossification, and 1 foetus with micrognathia at the top dose of 500 mg/kg bw/d.  This 1 case of micrognathia was considered by RAC to possibly be related to the findings of the preceding study.  The dam with the affected foetus did not show higher toxicity than other dams of this group.  According to the historical control data provided in the study report (time span not specified), there was 1 foetus with agnathia (mandibula) and 1 with cheilognathopalatoschisis (cleft lip, palate and maxilla) among 6292 control foetuses.  The HCD incidence appears to be exceeded at least for mandibular malformations (micrognathia, agnathia) with 3 litters in two consecutive studies containing affected foetuses.  Overall, the micrognathia observed in both studies raised concern about developmental toxicity.  It was noted by RAC that that the litter incidences were relatively low and the effect occurred in presence of some maternal toxicity (reduced food consumption, haematotoxicity).

Supporting rat study: M-493963-01-1, 2003

No maternal toxicity was observed at the top dose of 500 mg/kg bw/d, although some haematotoxicity can be assumed at this dose based on the results of other studies.  The choice of the top dose was based on results of a range-finding study where 1000 mg/kg bw/d reportedly caused lethargy, reduced food consumption and macroscopic findings (1-2 out of 5 animals with lung and kidney congestion and mottled liver).  Infact of the liver was observed at 0, (5 fetuses (5 litters)), 10 (6(6)), 100 (19*(10)) and 100 mg/kg bw/d (17*(8)).  The concern about infarct of the liver was considered by RAC to have been notably reduced by the relatively high incidence in concurrent controls.  The observed skeletal anomalies, although seen in the absence of maternal toxicity, were considered to be of low toxicological significance.  There was no effect on foetal body weight.  Overall, the developmental findings in this study were not considered by RAC to be sufficient for classification.

Supporting rat study: M-146990-01-1, 1991

The main manifestations of maternal toxicity in this study at the top dose level of 1000 mg/kg bw/d were effects related to haemolytic anaemia.  Most animals showed discoloured urine (indication of haemoglobinuria and/or haemosiderinuria) on one or more days of gestation and splenic weights were markedly increased (by 80%).  The effects on maternal body weight were limited (corrected maternal bw reduced by 3%).  Foetal weight at the top dose was reduced by 20%.  The reduction in mean foetal weight did not correlate with maternal corrected body weight gain at the level of individual animal data.  Several anomalies were observed at a low incidence at the top dose that were not present in
controls or at lower doses. The most notable ones were interventricular septal defect (3 foetuses in 2 litters) and cleft palate (3 foetuses in 1 litter). One of the foetuses with interventricular septal defect had additionally enlarged right atrium and reduced right ventricle. Two cases of
interventricular septal defect occurred in a litter with a high post-implantation loss.  The dams with the affected foetuses did not show higher general toxicity compared to other dams of this group.  Published HCD reported an average foetal incidence of ~1:6000 for ventricular septal defect and ~1:10000 for cleft palate.  This indicates that the malformations are relatively rare.  In addition, 2 foetuses from 1 litter showed partial duplication of inferior vena cava. However, as occurrence of this anomaly was limited to one litter and was not seen in three other rat PNDT studies, it was considered by RAC to be of less concern than the other malformations that were observed in multiple litters or studies.  Overall, reduced foetal weights and a low incidence of malformations were observed at the top dose in this study where maternal animals suffered from anaemia.  Although the maternal anaemia might have contributed to the developmental toxicity, the relationship between maternal and developmental toxicity has not been unequivocally demonstrated.  Therefore, interventricular septal defect and cleft palate were considered to be relevant for classification.

Key rabbit study: M-146988-01-1, 1991

The top dose level of 270 mg/kg bw/d in this study was based on a dose-range finding study where a dose level of 360 mg/kg bw/d caused abortion in 3 out of 7 dams.  Five out of 7 dams (including those aborting) in the range-finding study showed markedly reduced food  consumption (~60% GD 7-16) and an associated reduction in body weight gain.  In view of the marked maternal toxicity at 360 mg/kg bw/d, the top dose in the main study (270 mg/kg bw/d) was considered by RAC to be sufficiently high.  Food consumption at the top dose in the main study was reduced by ~50% in the dosing period, which still indicates significant maternal toxicity.  Corrected body weight was reduced by 7%; however, corrected body weigght is not a suitable indicator of toxicity in this case due to the long interval between the end of dosing (GD 18) and sacrifice (GD 29).  An increased incidence of early embryonic deaths and slight caudal pelvic shift was observed not only at the maternally toxic top dose level, but also at the mid-dose level, where maternal toxicity was minimal.  As the increase in early embryonic deaths at the mid-dose of 90 mg/kg bw/d was not marked (2.6-fold) and slight caudal pelvic shift is not considered a malformation, concerns about the developmental findings from this study alone are not sufficient to trigger classification. However, the early resorptions can be used as additional support for classification triggered by other effects.

Supporting rabbit study: M-146731-01-1, 1984

The choice the top dose level in this study (450 mg/kg bw/d) was based on a dose-range finding study, the results of which are not presented in the main study report.  Maternal animals at the top showed reduced food consumption (by 26%).  Terminal body weight corrected for gravid uterus weight was reduced by 5%.  Two abortions that occurred on GD 27-28 in the top dose group were considered likely to be a manifestation of maternal toxicity.  No increase in malformations was observed in this study.  Foetal body weight was markedly reduced at the top dose level (by 31%, not fully explained by the concurrent maternal toxicity) and there was also a slight increase in post-implantation loss without a clear dose-response relationship.

 

Justification for classification or non-classification

The classification of desmedipham for reproductive toxicity was considered by RAC (2019) based on the same dataset.  The conclusions of RAC are summarised below.

Conclusion on the classification for fertility and sexual function
Several effects potentially related to fertility and sexual function were identified in the available multigenerational studies: reduced epididymal sperm count, delayed puberty onset and reduced litter size. The reductions in litter size were not considered sufficient for classification due to the small size of the effect. The observed reduction in epididymal sperm count and the delayed puberty onset might represent a borderline case for classification.  But, given the magnitude of the effects and the other factors reducing the concern, RAC concluded that no classification for adverse effects on sexual function and fertility was justified.

Conclusion on classification for development
Several malformations of high concern (micrognathia, cleft palate, interventricular septal defect) were observed at low incidences in the rat PNDT studies at dose levels associated with maternal anaemia.  Although it could not be excluded that maternal toxicity contributed to these effects, unequivocal evidence for a causal relationship between maternal and developmental toxicity was missing.  The occurrence of malformations can in principle lead to classification in Category 1B. However, taking into account the low incidences and the evidence of concurrent maternal toxicity, RAC concluded to classify desmedipham in Category 2 (H361d).

Conclusion on classification for lactation
The reductions in pup body weight attributable to lactation seen in the generational studies with desmedipham were not considered by RAC to be of sufficient magnitude to warrant classification, or there were other factors reducing the concern (maternal toxicity, transient nature of the reduction).  Therefore, RAC concluded no classification for effects on or via lactation

Given the recent consideration of desmedipham by RAC based on the same dataset, no change to the harmonised classification is proposed.  Desmedipham is classified in Category 2 for effects on development (H361d).