3-(2-dodecenyl)succinic anhydride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000.

Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dihydro-3-(tripropenyl)furan-2,5-dione

EC Number:

295-556-6

EC Name:

Dihydro-3-(tripropenyl)furan-2,5-dione

Cas Number:

92077-08-2

IUPAC Name:

92077-08-2

Test material form:

other: liquid

Details on test material:

Name: Tripropenyl succinic anhydride
CAS No.: 92077-08-2
Batch No.: ESD0010823
Chemical Name: dihydro-3-(tripropenyl)furan-2,5-dione
pH: 1-2
Physical State at RT: liquid
Density: 1.01 g/cm3 (20°C, DIN 51757)
Colour: yellow to brown
Expiry Date: 01.12.2012
Storage Conditions: at room temperatureS
afety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: Males and non-pregnant nulliparous females
Age at the beginning of the study: 10-11 weeks old
Body weight at the beginning of the study: interval within ± 20% of the mean weight.
The range of the body weight was:
Females: 173-200g, (mean: 189.18 ± 20%= 37.84 g)
Males: 290-325g, (mean: 303.83 g, ± 20%= 60.77 g)

Number of animals: 10 Males and 10 females per group
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

Housing and Feeding Conditions:
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions:
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.0906)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
-housed individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (Lot No.030512)
Certificates of food, water and bedding are filed at BSL BIOSERVICE.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 4 mL / kg body weight.

For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Details on mating procedure:

Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the nominal concentration of test item in dosing formulations, samples were retained from all groups in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) - and stored between -15 and -35 °C (total 16 samples).

Dose formulations were not sampled for stability analysis at (0 hr) and 6 hours after preparation, as stability analysis was performed in 28 day repeated dose toxicity study with Tripropenyl Succinic anhydride.

In the first week and fifth week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, mid and low dose formulations and stored between -15 and -35 °C (total 18 samples)

At the end of the treatment period, all samples of dosing formulations were shipped on dry ice and protected from light to IBACON GmbH, D-64380 Rossdorf, Germany

The determination of test item concentration in the dosing formulations was performed by IBACON GmbH, in accordance with GLP.

Duration of treatment / exposure:

Males- 28 days
Females- Approximately 54 days

Frequency of treatment:

7 days/week

Details on study schedule:

Date of Draft Study Plan: 29 June 2012
Date of Final Study Plan: 04 July 2012
Date of 1st amendment 12 September 2012
to study plan
Date of 2nd amendment 17 September 2012
to study plan
Date of 3rd amendment 27 September 2012
to study plan

Date of Start of Experiment 05 July 2012
Date of End of Experiment: 03 Setpember 2012
Date of Draft Report (BSL): 28 January 2013
Date of Final Report (BSL): February 2013

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150, 250 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

10 animals/sex/group

Details on study design:

Number and Sex of the Animals:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). The study included three dose groups (LD, MD and HD) and one control group (C).

Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Experimental group and Dosage:
In consultation with the sponsor and based on the a BSL dose range finding study, doses levels of 50, 150, 250 were selected for the 3 dose groups (LD, MD and HD) and 1 control group (C)

The animals in the control group were handled in an identical manner to the dose group subjects and received corn oil in the same volume as used for treatment groups.

Administration of Doses:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 4 mL / kg body weight.
For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
Animals were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality. pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.

Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In the male animals, the body weight was taken weekly during the entire study period and on day of terminal sacrifice.
In the female animals the body weight were taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.

Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period and post mating period in males.

Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Gross Pathology:
Males were sacrificed after the completion of mating period (total dosing of 28 days) and females were sacrificed on respective post natal day 4 along with pups by using high dose of Ketamine/Xylazine (2:1). At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Pups sacrificed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.

The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole) were preserved in 10 % neutral buffered formalin. Testes and epididymides were initially preserved in modified Davidson’s Solution for approximately 24 hours and transferred to 10 % neutral buffered formalin.

Additional organs of animals which died during the course of the study were preserved and examined histopathologically in order to determine the cause of death.

Organ Weight:
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female and kidneys from few adult females were weighed.
Paired organs were weighed separately. Organ weights of animals found dead were not taken.

Histopathology:
A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the control and high dose groups which are sacrificed at the end of the treatment period.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS- Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Positive control:

Not included

Examinations

Parental animals: Observations and examinations:

Males and Females: Body Weight, Food Consumption, Clinical Signs, Mating behaviour, organ weight, Gross pathology and Histopathology.

Oestrous cyclicity (parental animals):

Not evaluated

Sperm parameters (parental animals):

Not evaluated

Litter observations:

Number and sex of pups, still births, runts, gross abnormalities of the pups and litter weight

Postmortem examinations (parental animals):

Males- Day 29; Females: On PND 4

Postmortem examinations (offspring):

Not examined (only gross external findings)

Statistics:

A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

Copulation Index (%), Fertility Index (%) and Delivery Index (%)

Offspring viability indices:

Viability Index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

Mortality:
One male animal (21) from MD group was found dead on premating day 6.
In females, 8 animals (71, 72, 73, 74, 75, 77, 78 and 80) from the HD group died on premating day 5, 5, 6, gestation day 11, 21, 22, 23 and 21, respectively.

Clinical Observation:
Test item related clinical signs were observed in male and female MD and HD group animals. Predominant clinical signs observed in male animals were slight salivation (3/10 in MD and 9/10 in HD), moderate salivation (2/10 in HD), slight piloerection (4/10 in MD and 6/10 in HD), moderate piloerection (2/10 in MD and 1/10 in HD), moving the bedding (7/10 in MD and 10/10 in HD) and abnormal breathing (1/10 in HD).

In females, predominant clinical signs observed were slight salivation (2/10 in LD, 6/10 in MD and HD), moderate salivation (4/10 in MD and HD), slight piloerection (1/10 in C, 6/10 in MD and HD), moderate piloerection (1/10 in MD and 3/10 in HD), moving the bedding (9/10 in MD and 6/10 in HD) and clonic convulsion and tremor (1/10 in HD).

Body Weight and Body Weight Change:
In males, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups. However, biologically significant decrease in body weight and bodyweight change was observed in MD and HD group when compared with controls.

In females, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups. However, a biologically significant decrease in body weight and bodyweight change was observed from gestation day 20 to postnatal day 4 in MD and HD group when compared with controls.

Food Consumption:
In males, statistically significant decrease in food consumption during premating day 1-7 and biologically significant decrease during premating day 7-14 was observed in MD and HD group when compared with controls. This effect on male food consumption could be considered as treatment related.

In females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.


Gross Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males. The few spontaneous gross pathological findings observed in male animals were yellow spot on right epididymides (1/10 in MD males) and yellow spot on left epididymides (1/10 in HD males).

In females, predominant gross pathological findings observed in dead animals were white area on left liver lobe (1/10 in HD females), discoloured stomach (3/10 in HD females), gaseous distension of intestinal tract (3/10 in HD females), dark spot on lung (1/10 in HD females), discoloured red thymus (2/10 in HD females) and small thymus (1/10 in HD females)

These gross pathological findings observed in males were spontaneous in nature. However, the significance of these macroscopic findings in dead females remains unclear and an influence of beginning of autolysis cannot be excluded.

Organ Weight:
In males, statistical analysis of organ weight data revealed a significant decrease in absolute prostate and seminal vesicles with coagulating gland weights in HD group when compared with controls.

In females, a statistically significant increase in relative kidney weights (left and total) was observed in MD and HD group when compared with controls.

Since in males, decrease in absolute prostate and seminal vesicles with coagulating gland weights was minimal and no effect on relative weight was observed, this effect on male weights was considered to be non adverse. However, in the light of histopathological findings in the kidneys, effect on kidney weights in females was considered to be test item related.

Histopathology
One male rat from the MD group and eight females from the HD group were found dead during the treatment period. Gavage accident was established as direct cause of death for one decedent high dose female. In addition, in all decedents combinations of histopathological findings were noted in several of the following organs: kidney, forestomach, lymphoid organs (spleen, thymus, Peyer's patch, mesenteric and axillary lymph node), lung, adrenal gland and heart, and were considered to have contributed to the death of these animals.

Renal tubular degeneration/regeneration was considered to be possibly directly test item-related. In the forestomach, minor (sub)mucosal mixed cell infiltration, submucosal congestion/hemorrhage and (peri)vasculitis were also considered to represent direct effects of the test item. For lymphoid atrophy and single cell death of lymphoid organs, as well as for leukocytostasis and vasculitis/thrombi in the lung, a direct test item relationship was not clear. Adrenal gland changes (cortical degeneration/necrosis and diffuse cortical hypertrophy) and heart changes (cardiomyocyte vacuolation, focal myocardial degeneration) were considered to be secondary.

At terminal sacrifice, the kidney was evaluated in all surviving females. Against a background incidence noted in control females, minimal to moderate numbers of the basophilic (regenerative) tubules in the cortex and medulla occurred in the females of all three dose groups, without a clear dose relationship. Renal changes were considered to be possibly directly test item-related.

Altogether, there was no evidence of a direct test item-related effect on male or female reproductive organs in this study.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: signs of toxicity in both male and females and mortalities in 250 mg/kg body weight group females and 150 mg/kg Body weight group males.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Only gross external observations were made

Histopathological findings:

not examined

Details on results (F1)

Litter Weight Data:
No statistically significant effect on litter weight data was observed in treatment groups when compared with controls. However, biologically significant decrease in group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 was observed in MD and HD group when compared with controls.

Precoital Interval and Duration of Gestation:
No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.

Successful mating resulted in 10, 8, 9 and 3 pregnancies in the control, low, mid and high dose respectively.

Pre and Post Natal Data:
Pre and post-natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls. However, there was a biologically significant increase in post implantation loss observed in MD group although statistical significance was not achieved. This increase in group mean post implantation loss in MD group could be attributed to the very low number of live pups born and high difference in implantation sites and number of pups born on PND 0 from two MD females (66 and 69). Group mean values from remaining 7 females were comparable to the controls and therefore this increase in post implantation loss in HD group was considered to be non adverse and within the range of biological variation.

Litter Data:
Statistical analysis of litter data revealed no treatment related effect on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.

All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls except high numbers of still births (11) were observed in one female (66) from MD group.

Reproductive Indices:
No treatment related effect on copulation index and delivery index was observed when compared with controls, However, a reduced fertility index (No. of pregnant females/No. of copulated females X 100) in HD (42.9) and viability index in MD (86.87) was observed as compared to control and LD groups.

Pup Survival Data:
Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study. However, 1 pup from LD group- female 56 (pup no. 9), 2 pups from MD group female 64 (pup no. 3 and 6), female 66 (pup no. 1 and 2) were missing and presumed to be cannibalized by the dam between PND 0-2. Since this incidence of cannibalism was observed in just 3 females and it was within the rat cannibalism rate, this incidence was not considered to be test item related.

Pup External Finding:

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of developmental toxicity in pups up to 250 mg/kg Body weight

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Repeated dose administration of tripropenyl succinic anhydride in corn oil to ten male (28 days) and 10 female (maximum 54 days) Wistar rats at dosages of 50, 150 and 250 mg/kg body weight revealed mortality but no significant findings of toxicological relevance in HD group females. Data can be read-across among members of the C8-12 Alkenyl Succinic Anhydrides Category, based on common functional groups, similar break-down products and potency patterns among carbon-chain length. This is adequate to fulfill the information requirements, to be the basis for classification and labelling decisions, and for risk assessment.

Based on the data generated from this reproduction/ developmental toxicity screening test with Tripropenyl Succinic anhydride, the no observed adverse effect level (NOAEL) for parental animals was considered to be 50 mg/kg body weight and NOAEL for developmental toxicity of pup was believed to be 250 mg/kg body weight.

Executive summary:

The aim of this study was to assess the possible effect of Tripropenyl Succinic Anhydride on male, female fertility and embryofetal development in Wistar rats .This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with 50, 150 and 250 mg/kg body weight per day of Tripropenyl Succinic anhydride at dose volume of 4 mL/kg body weight. The test item was formulated in corn oil. Control animals were handled identically as treated groups and received corn oil in similar volume as treated groups.

The doses evaluated were  0, 50, 150 and 250  mg/kg body weight/day

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for total period of 28 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period and post mating period in males where food consumption was not measured.

After 14 days of premating treatment to both male and female, animals were paired (1:1) for 14 days. The subsequent morning onwards, the vaginal smears of females were checked to confirm the evidence of mating in the form of sperm positive vaginal smears. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition and on day 4 post-partum.

All surviving male and females were sacrificed on day 29 and post natal day 4 respectively and subjected to necropsy. Copulated but non pregnant female (two, No. 54 and 62) were sacrificed on respective day 26 after the evidence of mating.  The wet weight of male and female reproductive organs and kidney weights of few females was taken and preserved in 10 % neutral buffered formalin except testes and epididymides which were initially fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin.

Histopathological evaluation of the tissues was performed on reproductive organs and macroscopic findings of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Female reproductive organs were additionally evaluated in medium dose group females as there was high mortality in high dose group. Kidneys of few terminally sacrificed females from all groups were evaluated histopathologically. A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle. In decedents, set of the organs and tissues were evaluated to identify cause of the death. Organs showing gross alterations were also examined histopathologically.

For determination of the nominal concentration of test item in dosing formulations, samples were retained from all groups in study week 1, 3, 5 and 7. Dose formulations were not sampled for stability analysis at (0 hr) and 6 hours after preparation as stability analysis was performed in 28 day repeated dose toxicity study withTripropenyl Succinic anhydride.

In the first week and fifth week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, mid and low dose formulations and all samples were stored between -15 and -35 °C until analysis.

Summary Results

Mortality

One male animal from MD group was found dead on premating day 6 and 8 females (71, 72, 73, 74, 75, 77, 78 and 80) from HD group were died on premating day 5, 5, 6, Gestation day 11, 21, 22, 23 and 21, respectively.

Clinical Signs

Test item related clinical signs were observed in male and female MD and HD group animals. Predominant clinical signs observed in male animals were slight salivation, moderate salivation, slight piloerection, moderate piloerection, moving the bedding and abnormal breathing.

In females, predominant clinical signs observed were slight salivation, moderate salivation, slight piloerection, moderate piloerection, moving the bedding and clonic convulsion and tremor.

Body Weight Development

In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups. However, biologically significant decrease in body weight and bodyweight change was observed in MD and HD group when compared with controls. 

In females, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups. However, biologically significant decrease in body weight and bodyweight change was observed from gestation day 20 to postnatal day 4 in MD and HD group when compared with controls. 

This effect on male and female body weight development could be considered as treatment related.

Food Consumption

In males, statistically significant decrease in food consumption during premating day 1-7 and biologically significant decrease during premating day 7-14 was observed in MD and HD group when compared with controls. This effect on male food consumption could be considered as treatment related.

In females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

 Litter Weight Data

No statistically significant effect on litter weight data was observed in treatment groups when compared with controls. However, biologically significant decrease in group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 was observed in MD and HD group when compared with controls.

Precoital Interval and Duration of Gestation

No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data

Pre and post-natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent pre-implantation and post-implantation loss remained unaffected due to treatment when compared with controls. However, there was biologically significant increase in post implantation loss observed in MD group although statistical significance was not achieved. However, this effect was attributed to higher values from two females and group mean values from remaining 7 females were comparable to the controls and therefore this increase in post-implantation loss in HD group was considered to be non adverse and within the range of biological variation.

Litter Data

Statistical analysis of litter data revealed no treatment related effect on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.

All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls except high numbers of still births (11) were observed in one female (66) from MD group.

Reproductive Indices

No treatment related effect on copulation index and delivery index was observed when compared with controls, however, reduced fertility index in HD (42.9) and viability index in MD (86.87) was observed as compared to control and LD groups.

Pup Survival Data

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study. However, 1 pup from LD group, 4 pups from MD group were missing and presumed to be cannibalized by the dam between PND 0-2. Since this incidence of cannibalism was observed in just 3 females and it was within the rat cannibalism rate, this incidence was not considered to be test item related.

Pup External Findings

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, in control and LD females, one pup each was observed for dark nose on PND 0. This finding considered to be spontaneous in nature and not related to the treatment with test item.

Gross Pathology

At necropsy of male, macroscopic examination of the animals revealed no test item related macroscopic findings in males. Few spontaneous gross pathological findings observed in 2 male animals were yellow spot on right epididymides and yellow spot on left epididymides.

In females,predominant gross pathological findings observed in dead animals were white area on left liver lobe, discoloured stomach, gaseous distension of intestinal tract, dark spot on lung, discoloured red thymus and small thymus.

These gross pathological findings observed in males were spontaneous in nature However,the significance of these macroscopic findings in dead females remains unclear and an influence of beginning of autolysis cannot be excluded.

Organ Weight

In males, statistical analysis of organ weight data revealed significant decrease in absolute prostate and seminal vesicles with coagulating gland weights in HD group when compared with controls

In females, statistically significant increase in relative kidney weights (left and total) was observed in MD and HD group when compared with controls.

Since in males, decrease in absolute prostate and seminal vesicles with coagulating gland weights was minimal and no effect on relative weight was observed, this effect on male weights was considered to be non adverse. However, in the light of histopathological findings in the kidneys, effect on kidney weights in females was considered to be test item related.  

 

Histopathology

In all decedents combinations of histopathological findings were noted in several organs like kidney, forestomach, lymphoid organs, lung, adrenal gland and heart, and were considered to have contributed to the death of these animals.

Renal tubular degeneration/regeneration was considered to be possibly directly test item-related. In the forestomach, minor (sub)mucosal mixed cell infiltration, submucosal congestion/hemorrhage and (peri)vasculitis were also considered to represent direct effects of the test item. For lymphoid atrophy and single cell death of lymphoid organs, as well as for leukocytostasis and vasculitis/thrombi in the lung, a direct test item relationship was not clear. Adrenal gland changes (cortical degeneration/necrosis and diffuse cortical hypertrophy) and heart changes (cardiomyocyte vacuolation, focal myocardial degeneration) were considered to be secondary.

At terminal sacrifice, kidney was evaluated in all surviving females. Against a background incidence noted in control females, minimal to moderate numbers of basophilic (regenerative) tubules in the cortex and medulla occurred in the females of all three dose groups, without a clear dose relationship. Renal changes were considered to be possibly directly test item-related.

Altogether, there was no evidence of a direct test item-related effect on male or female reproductive organs in this study.