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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021 - 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-bis(2,3-epoxypropoxy)butane
EC Number:
219-371-7
EC Name:
1,4-bis(2,3-epoxypropoxy)butane
Cas Number:
2425-79-8
Molecular formula:
not applicable, UVCB
IUPAC Name:
N,N-dimethylacetamide
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Source/Supplier: Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal,, RR Dist, Telangana 500078, India Bred under barrier conditioned
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Sprague-Dawley rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Acclimatization: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before the pre-treatment period. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that were determined by the veterinarian to be suitable for use were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.
From 45 male and 50 female animals used for acclimatization 4 groups, 20 rats (10 males + 10 females) per group were assigned as follows: Vehicle control (G1), Low dose (G2), Mid dose (G3), and High dose (G4) in this study in total 80 animals, 40 males and 40 females.
Age of animals at the start of treatment was 13 weeks and the body weight ranged from 384.06 to 461.85 g for males and 252.67 to 292.21g for females; at the commencement of the treatment, the weight variation of rats used did not exceed ±20 % of the mean body weight in each sex and group.
Mean body weight at the start of treatment (g) for males and females:
G1: 434.83± 19.49 (m); 274.70 ± 13.79 (f)
G2: 435.15 ± 25.15 (m); 274.70 ± 11.85 (f)
G3: 432.79 ± 28.65(m); 276.36 ± 12.07 (f)
G4: 431.59± 22.63(m); 275.11 ± 11.34 (f)
Temporary identification of all animals was done by using a cage card and crystal violet solution body marking during acclimatization and pre-treatment periods. Permanent identification of all rats was ensured using a rat accession number, cage card and tail tattooing with the numerical details of the rat accession number only (Tail tattooing was done on Day 2 of treatment). Turmeric body marking was done on the day of grouping during the pre-treatment period for permanent identification and animals were not re-marked thereafter. Pups were identified using the nail clipping method.
Rats were housed in an environment-controlled room. The temperature maintained during the experiment was between 19.1 and 24.4 °C and relative humidity between 55 and 68 %. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.8 – 13.0 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
During pre-mating two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless-steel sipper tubes.
During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).
Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. These objects were provided during the pre-mating and post-mating period for males and during the pre-mating period for females and changed along with cage once a week.
Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.
Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum to the rats. A sample of the diet was retained and discarded prior to finalization of the report.
Deep bore-well water passed through an activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
The food and water provided to the animals were tested for contaminants. Analysis and contaminant analysis reports of the food and water are included in the final report.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Carboxymethyl cellulose (Medium viscosity) in Milli-Q water
Details on exposure:
The stability of the test item in the vehicle (0.5% Carboxymethyl cellulose (Medium viscosity) in Milli-Q water) was established at 1 and 50 mg/mL (Study No. G24244). The test item was also found to be stable in the vehicle for 24-hours at 1 mg/mL and up to 48-hours at 50 mg/mL at room temperature.
Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume) for each dose levels separately and a small volume of vehicle was added and mixed using a glass rod. The volume was made up to the mark using the vehicle to get the final desired concentration of 5, 20 and 50 mg/mL for the G2, G3, and G4 groups, respectively.
100 mL of the dose formulations were prepared as follows:
G2: Dose 50 (mg/kg bwt/day), Dose volume 10 mL/kg, Concentration 5 mg/mL, 100 mL Volume to be made up, 500 mg Test item weight
G3: Dose 200 (mg/kg bwt/day), Dose volume 10 mL/kg, Concentration 20 mg/mL, 100 mL Volume to be made up, 2000 mg Test item weight
G4: Dose 500 (mg/kg bwt/day), Dose volume 10 mL/kg, Concentration 50 mg/mL, 100 mL Volume to be made up, 5000 mg Test item weight
The dose formulations were prepared once daily. The formulations were constantly stirred using a magnetic stirrer during test item administration and during sampling. The volume of the dose formulations prepared varied depending on the requirement and/or body weights of the rats recorded during the experimental period.
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during the 2nd month (day 34) of treatment and analysed in-house. For each set, Duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Study No.: G24244. One set of samples was analysed for concentration (a.i) analysis and another set (second set) of samples was stored at ambient condition for reanalysis purpose as a backup. Formulations were considered acceptable if the overall mean results of all the layers were within the range 70.0 % to 120.0% and the overall relative standard deviation (% RSD) was equal to or less than 20.0 %.
Treatment
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 43 days at approximately the same time each day (varying by ±3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ±3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 40 - 60 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.
Details on mating procedure:
One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and/or vaginal plug. All the females were successfully copulated within ten days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they litter. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the study an analytical method for test substance quantification was developed and suitability to control doses applied was proven in a Dose Formulation Analysis Report (Study No. G24243). The test item in the dose formulations was determined using Gas Chromatography with FID Detector (GC).
Pure 1,4-bis(2,3-epoxypropoxy) butane was used as reference Standard, provided by the sponsor (Batch No: 2022717/007, Purity: 100% (UVCB) Expiry Date: 16 June 2023). With this reference standard the GC was calibrated. No interference of other signals with the target signal were noted.
For homogeneity and active ingredient concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during the 2nd month (day 34) of treatment and analysed in-house. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The samples from the dose formulations were diluted in such a way to get the final concentration of about 100 µg/mL of 1,4-bis(2,3-epoxypropoxy) butane. Similarly, the control (vehicle) sample was processed. Before injecting the prepared dose formulations sample to GC, it was quantitatively transferred through a funnel plugged with cotton and magnesium sulphate heptahydrate (to remove the moisture).
System suitability and standard check performed on the day of analysis were satisfactory (see original report for details).
The results of dose formulation analysis were considered acceptable, as the over all mean percent recovery was in the range, 70.0% to 120.0% of the claimed concentration at each dose level and over all %RSD at each dose level was less than or equal to 20%.
Duration of treatment / exposure:
To male rats the dose formulation was administered orally by gavage of the specific groups once daily for 43 days at approximately the same time each day (varying by ±3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Female rats received the dose formulations orally by gavage to the specific group once daily at approximately the same time each day (varying by ±3 hours). This included 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 40 - 60 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
Frequency of treatment:
Once daily, approximately the same time each day (varying by ±3 hours)
Details on study schedule:
Each dose group in this OECD 421 study with rats consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.
During the conduct of this study, the prepared dose formulations and vehicle were analyzed for test item concentration on Days 1 and 32 of treatment. The results indicated that the overall mean results of 1,4-bis(2,3-epoxypropoxy)butane in the tested formulation were in the range of 70 % to 120 % of the claimed concentration and the overall relative standard deviation (% RSD) were <20.0%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.
All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.
Histopathological examination was carried out on all the preserved organs and tissues of control (G1) and high dose (G4) group rats and on all gross lesions. Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
10 males and 10 females per dose group (including control)
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of Dose Levels and Dose Justification
Following summary data of a 28-Day Oral Gavage Toxicity Study conducted with 1,4-butanediol diglycidylether (BDDGE) and a Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the reproduction/developmental toxicity screening test conducted using the analogue substance 1,6-hexanediol diglycidylether (HDDGE) was provided by the Sponsor.
A 28-Day Oral Gavage Toxicity Study was conducted with 1,4-butanediol diglycidylether (BDDGE) in Sprague Dawley Rats dosed by oral gavage at 25, 100, 200 and 400 mg/kg in the vehicle, 0.5% w/v carboxymethylcellulose in deionized water (Study Number WIL-749003, 30 July 2010). The dose volume was 10 mL/kg for all groups. This study can be used as dose range finding information for the OECD 421 study. The NOAEL for systemic toxicity was 200 mg/kg/day. This level was based on the finding of several effects, including lower body weights (up to -14.6% in males and -6.4% in females) and body weight gains (males), as well as changes to haematological and clinical chemistry parameters and higher liver weights (both sexes). The NOAEL for local site of contact toxicity could not be established in males whereas in females, the NOAEL for local site of contact toxicity was considered to be 25 mg/kg/day (the lowest tested dose). There were no clear adverse target organ changes (changes in liver, pancreas, exorbital lacrimal gland were considered test-item related, but not clearly adverse). Stomach lesions (edema, granulation, inflammation, hyperplasia) were considered to be severe and appearing at low concentrations (starting at lowest dose formulation of 2.5 mg/mL, increasing at higher concentrations). The latter changes may have resulted in slightly lower body weights, although no changes in food consumption were reported. Based on the available data the dosing of 400 mg/kg was considered to be toxic in males and subtoxic in females. The toxicity can be secondary to stomach lesions which were present at all doses, but more explicit at 200 and 400 mg/kg bw/d.
A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the reproduction/developmental toxicity screening test was conducted using the analogue substance 1,6-hexanediol diglycidylether (HDDGE) in Sprague Dawley Rats (Study No. WIL-749002, 2010). Rats were dosed at 50, 200, and 500 mg/kg/day administered at a dosage volume of 5 mL/kg bw/day in the vehicle, 0.5% carboxymethylcellulose (medium viscosity) in deionized water. The NOAEL for systemic toxicity was 200 mg/kg bw/d. Test substance-related toxicity findings were noted in the 500 mg/kg/day group males and females at the post-dose observations and included salivation, salivation-related findings, and/or red material around the nose, mouth, and/or forelimbs. In the 500 mg/kg/day dosed males and females, a test substance-related mean body weight loss (up to -15.6% in males and -5.6% in females) and reduced mean food consumption were noted. A test substance-related, adverse histologic injury to the non-glandular stomach was evident in all 500 mg/kg bw/day group males and females, characterized by hyperkeratosis, hyperplasia, and in most animals, ulceration. Less severe hyperkeratosis and hyperplasia, without ulceration, were noted in the 200 mg/kg/day males and females. In addition, an adverse low mean lymphocyte count and low globulin concentration was noted in the 500 mg/kg bw/day group females and males, respectively, and non-adverse high glucose and cholesterol serum concentrations were noted in the 500 mg/kg bw/day females. In the 500 mg/kg bw/day group males, an adverse low mean final body weight was noted, and evidence of non-adverse organ-specific effects was noted in the liver, kidneys, and adrenals in the 200 and/or 500 mg/kg bw/day group males. The final body weights were unaffected in the females, but several organ weight parameters were high in the 500 mg/kg bw/day group females, including the adrenal/FBW ratio, absolute kidney and kidney/FBW ratio, absolute liver and liver/FBW ratio, absolute thyroids/parathyroids and thyroids/parathyroids/FBW ratio.
Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels. Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 50, 200, and 500 mg/kg bw/day groups were similar to the control group values. Mean pup body weights and body weight gains at all dosage levels were unaffected by dose administration. No test substance-related clinical findings were noted for the F1 pups and there were no remarkable macroscopic findings in the F1 pups at the scheduled necropsy at any dosage level.
Based on the above results, 200 mg/kg bw was selected for BDDGE as medium dose level, whereas 50 and 500 mg/kg bw are proposed as low and high dose levels for the present OECD 421 study with 1,4-butanediol diglycidylether (BDDGE). These doses should allow sufficient interval between dosages to demonstrate a dose response (including toxicity at the highest doses), and to find NOAELs for the parental/maternal systemic, reproductive and developmental toxicity endpoints.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
All rats were observed for morbidity and mortalities twice per day, once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays.
Each rat was observed for clinical signs once daily during the treatment period. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals. Detailed clinical examination was done prior to the treatment on Day 1 and 1 - 3 hour after dosing at weekly intervals thereafter (±1 day) during the course of the in-life for all rats. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).
Individual body weights of males were recorded on Day 1 and at weekly (±1 day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till mating confirmation with males. Furthermore, all dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.
The food consumption was measured at weekly intervals (±1 day) during treatment. Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation periods.
Oestrous cyclicity (parental animals):
Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular
4 - 5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.
Sperm parameters (parental animals):
not performed
Litter observations:
The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded. The litters were observed daily to note the number of alive, dead and cannibalized pups. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded. In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded. On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
After standardization, the individual pup body weight was recorded on Day 13 of lactation.
The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight. The number of nipples/areolae in male pups was counted on PND 13.
All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.
Postmortem examinations (parental animals):
Gross Necropsy: All adult animals and pups were examined macroscopically for any structural abnormalities or pathological changes. The adult animals were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia, subjected to detailed necropsy and findings were recorded. All the surviving pups were necropsied on lactation Day 13 and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death. The uterus of the single non pregnant rat (G1-Rab7446) was stained with Salewski stain to identify the post-implantation loss of the embryos. The number of implantation sites were recorded for all the dams.
Organ/Tissue Collection, Weighing and Preservation: On completion of gross pathology examination, the tissues and organs noted below were collected and weighed from all adult animals. The organ weight ratios (organ to body weight) as percentage of fasting body weight were determined and presented in the report. The paired organs were weighed together and combined weight was presented. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes, as follows:
- Gland, Adrenal: organ weight, collection & preservation, microscopic examination
- All gross lesions: collection & preservation, microscopic examination
- Epididymis: organ weight, collection & preservation, microscopic examination
- Ovary: organ weight, collection & preservation, microscopic examination
- Oviduct: collection & preservation, microscopic examination
- Stomach: collection & preservation, microscopic examination
- Thymus: organ weight, collection & preservation, microscopic examination
- Gland, Thyroid/ Gland, Parathroid (Weighed after formalin fixation): organ weight, collection & preservation, microscopic examination
- Uterus (weighed along with cervix): organ weight, collection & preservation, microscopic examination
- Cervix: collection & preservation, microscopic examination
- Vagina: collection & preservation, microscopic examination
- Gland, Prostate (prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands): organ weight, collection & preservation, microscopic examination
- Gland, Seminal vesicle/ Gland, Coagulating (prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands): organ weight, collection & preservation, microscopic examination
- Testis (collected in modified Davidson’s fluid): organ weight, collection & preservation, microscopic examination
- Muscle, Levator Ani/ Bulbospongiosus: organ weight, collection & preservation
- Gland, Bulbourethral: organ weight, collection & preservation
- Penis, Glans: organ weight, collection & preservation
On Day 13, thyroid gland from available one male and female pup from each litter (randomly selected) were collected and preserved in 10% NBF for the histopathological examination. The thyroid weight was determined after fixation.
Histopathology: Histopathological examination was carried out on all the preserved organs and tissues of control (G1) and high dose (G4) group rats and on all gross lesions Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The reproductive organs of a not littered female (G1: Rab7446) were examined.
Stomach in both sexes and thymus in females were suspected of showing test item-related histopathological changes in high dose (G4) group and hence examined in the lower dose (G2 and G3) groups.
The tissues were processed for routine paraffin embedding and 4 - 5 micron sections were stained with Haematoxylin Eosin stain. In addition, testes were sectioned at 3 - 4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues will be archived.
Postmortem examinations (offspring):
see above!
Statistics:
Data was captured using the Provantis™ laboratory information management system (LIMS). Parameters such as body weight, body weight change, food consumption, organ weights, organ weight ratios (organ to body weight), oestrous cycle, ano-genital distance, pre-coital interval, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (thyroid profile) data were evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be non-homogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like mating and fertility indices were analysed using Chi-square test. When Chi-square found significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in Provantis™ built-in statistical tests.
Data captured outside of Provantis™: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0.
For two groups, the comparisons of mean between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers were presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences were designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05
Reproductive indices:
Male mating index (%) = Number of males with evidence of mating x 100/ Number of males cohabited
Male fertility index (%) = Number of males siring a litter/impregnated a female x 100/ Number of males cohabited
Female mating index (%) = Number of females mated x 100/Number of females cohabited
Female fertility index (%) = Number of pregnant females x 100/Number of females used for mating
Mean number of implantations/groups = Total number of implantations/Total number of pregnant animals
Post implantation loss (%) = Number of implantations - Number of live x 100/Number of implantations
Offspring viability indices:
Mean litter size per group = Total Number of pups born/Total Number of littered animals
Mean viable litter size = No. of viable pups/No. of females littered
Live birth index (%) = No. of viable pups born (at first observation) x 100/Total no. of pups born (at first observation)
Day 4 survival index (%) = Number of viable pups on lactation Day 4 x 100/Number of viable pups born
Sex Ratio (%) = No. of male pups born x 100/Total no. of pups born
Ano-genital Distance Ratio (mm/g1/3 ) = Ano-genital distance/Cube root of body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs or mortalities observed throughout the treatment period in either sex at 50 mg/kg/day. At 200 mg/kg/day, transient clinical sign of slight salivation was observed in 4 males and 6 females soon after the dose administration in few animals from treatment Day 41. One female (Rab7490) showed sparse hair loss during days 53 to 54 of treatment. At 500 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 32. Two females (Rab7503 and Rab7506) showed sparse hair loss during days 52 to 55 of treatment.
Nevertheless, the symptom of transient clinical sign of slight salivation was subsided within a few minutes and the rats were found to be normal. The incidence of sparse hair loss observed were common in rodents and considered incidental findings.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
See also clinical signs above!
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: At 500 mg/kg bw/day, the mean body weights were significantly lower (p < 0.05) throughout the treatment period with the reduction of 10.17 to 15.58%, compared to vehicle control group. The body weight gains during Days 1 - 43 were also significantly lower, compared to vehicle control group (reduction of 68.84%). The mean body weights and absolute body weight gains were unaffected by the treatment at 50 and 200 mg/kg bw/day doses tested, comparable to vehicle control group.
Females: The mean body weights were significantly lower (p < 0.05) on Day 15 with the reduction of 10.16% at 500 mg/kg bw/day. The absolute body weight gains during Days 1 - 15 were also significantly lower (reduction of 10.16%), compared to vehicle control group. The mean body weights and absolute body weight gains were unaffected by the treatment at 50 and 200 mg/kg bw/day the doses tested, comparable to vehicle control group.
The lower mean body weight and absolute weight gains observed in males at 500 mg/kg bw/day males and females were considered to be attributable to test item-related decrease in food consumption.
During gestation period at 500 mg/kg bw/day, the significantly lower (p < 0.05) mean body weights were observed on GD 20 with the reduction of 9.29%, compared to vehicle control group. The absolute body weight gain was also lower (statistically not significant) during GD 14 - 20 (reduction of 40.21%) and 0 - 20 (reduction of 18.74%), compared to the vehicle control group.
Treatment had no effects on the body weights and food consumption measured during different intervals of the gestation period at 50 and 200 mg/kg bw/day and food consumption at 500 mg/kg/day, when compared to vehicle control.
During lactation at 500 mg/kg bw/day, the significantly lower (p < 0.05) mean body weights were observed on Lactation Days 4 and 13 with the reduction of 10.31 to 11.81%. The absolute body weight gains were lower (statistically not significant) during LDs 0 - 4 (reduction of 54.50%), 4 - 13 (reduction of 57.50%) and 0 - 13 (reduction of 56.08%), compared to vehicle control group.
Treatment had no effects on the maternal body weight and body weight gain during lactation period at 50 and 200 mg/kg bw/day doses when compared to the vehicle control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: At 200 and 500 mg/kg bw/day, the food consumption was significantly lower (p < 0.05) during Days 1 - 8, 8 - 15, 29 - 36 and 36 - 43 with the reduction of 5.09 to 12.97 % and 16.91 to 31.66 % respectively, when compared to vehicle control group.
Females: At 500 mg/kg bw/day, the food consumption was significantly lower (p < 0.05) during Days 1 - 8 with the reduction of 20.03 % when compared to vehicle control group. The mean food consumption was significantly lower (p < 0.05) during days 1 - 8 & 8 - 15 with the reduction of 5.48 to 6.28% at 50 mg/kg bw/day.
The test item related decrease in food consumption was observed at 500 mg/kg bw/day males and females, associated with the decrease in the mean body weight and absolute body weight gains. These sporadic incidences of significant difference observed in food consumption at 200 mg/kg bw/day males and 50 mg/kg bw/day in females were toxicologically not significant as there was no dose dependency. Further, the overall mean body weights and absolute body weight gains were unaffected by the treatment.
The total and daily food consumption was significantly lower during LDs 0 - 4, 4 - 13 and 0 - 13 at 500 mg/kg bw/day with reduction of 15.20 to 29.68 %, associated with the decrease in the mean body weight and absolute weight gains.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Immunological findings:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during the treatment period, prior to cohabitation with males. The calculated mean oestrous cycle length was 4.0, 4.2, 4.0 and 4.1 days in vehicle control, low, mid and high dose groups, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
The vaginal smear was examined for all the animals prior to necropsy. The numbers of rats observed in the various stages of the oestrous cycle are detailed in the table below; no significant differences between dose groups and control were observed.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the mating and fertility indices of sires and dams at all the doses tested.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
presumably yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no abnormalities observed in pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The weight of male pups (16%) and total litter mean pups (15%) were significantly lower (p < 0.05) on Day 13 at 500 mg/kg bw/day, compared to vehicle control group.
At 200 mg/kg bw/day, the mean body weight of female pups (8 %) was significantly higher (p < 0.05) on Day 13 and considered as toxicologically not significant due to lack of dose response. The weight of male and female pups per litter and total litter mean pup body weight were not affected by the treatment at 50 and 200 mg/kg bw/day doses.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in thyroid hormone profile (TSH and T4) in adult animals and pups at all the dose levels.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No changes attributable to the test item were detected in the ano-genital distance, ano-genital ratio and ano-genital index in either sex.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The male pups did not exhibit areolae/nipple retention on PND 13 at any of the doses tested.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no external abnormalities in live or dead pups in any of the groups.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Treatment decreased (p < 0.05) the mean litter size and mean viable litter size on lactation days 0, 1 and 4, compared to vehicle control group. The number of pups culled on Day 4 was significantly lower (p < 0.05) at 500 mg/kg bw/day, as there was a lower mean litter size in this group, compared to the vehicle control group.
Test item had no treatment-related effects on the mean litter size, viable litter size and number of dead pups at first observation at 50 and 200 mg/kg bw/day.
There were no external abnormalities in live or dead pups in any of the groups.
The number of implantations were significantly lower (24%: 12.50 versus 16.44 in vehicle group; p<0.05) and post-implantation loss was higher (16.9% versus 5.3 in vehicle group, statistically not significant) at 500 mg/kg bw/day, compared to vehicle control group. No test item-related changes were observed in the number of implantations and percentage of post implantation loss at 50 and 200 mg/kg bw/day doses tested.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: At high dose group reduction in mean weight of pups and their organ weight is presumably attributable to maternal toxicity at the 500 mg/kg bw/d dose.

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
other: unspecific
Organ:
other: general toxicity such as reduced weight and organ weights
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
presumably yes

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

The numbers of rats observed in the various stages of the oestrous cycle prior to necropsy are detailed in the table below:












































Stages of Oestrous cycle


 



Group & Dose (mg/kg bw/day)



G1


0



G2


50



G3


200



G4


500



Proestrous



1



0



1



2



Oeestrous



0



0



0



0



Metestrous



0



0



0



0



Diestrous



9



10



10



8



 


Summary of Clinical Signs – Males
































































Observation Type: All Types


From Day 1 (Start Date) to 44 (Start Date)



Male



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Normal



 



 



 



 



Number of Animals Affected



10



10



10



10



First to Last seen



1 – 44



1 – 44



1 – 44



1 – 44



Salivation



 



 



 



 



Number of Animals Affected



0



0



4



10



First to Last seen







41 – 43



32 ‑ 43



 


Summary of Clinical Signs – Females










































































































Observation Type: All Types


From Day 1 (Start Date) to 61 (Start Date)



Female



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Normal



 



 



 



 



Number of Animals Affected



10



10



10



10



First to Last seen



1 – 60



1 – 60



1 – 60



1 – 61



Salivation



 



 



 



 



Number of Animals Affected



0



0



6



10



First to Last seen







41 – 58



32 – 60



Skin and Fur



 



 



 



 



Number of Animals Affected



0



0



1



2



First to Last seen







53 – 54



52 – 55



Not littered



 



 



 



 



Number of Animals Affected



1



0



0



0



First to Last seen



43 – 43









 


Summary of Body Weights – Males





























































































































































































































































Sex: Male


Day(s) Relative to Start Date



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Body Weight (g)



1 [a]



Mean



434.83



435.15



432.79



431.59



SD



19.49



25.15



28.65



22.63



N



10



10



10



10



%Diff



-



0.07



‑0.47



‑0.74



8 [a]



Mean



456.07



450.55



443.29



407.39*



SD



20.77



25.06



29.67



23.72



N



10



10



10



10



%Diff



-



‑1.21



‑2.80



‑10.67



15 [a]



Mean



490.90



479.86



474.12



420.07*



SD



20.45



31.74



35.88



19.74



N



10



10



10



10



%Diff



-



‑2.25



‑3.42



‑14.43



22 [a]



Mean



507.77



490.22



484.14



437.98*



SD



19.21



31.17



38.12



28.18



N



10



10



10



10



%Diff



-



‑3.46



‑4.65



‑13.75



29 [a]



Mean



520.25



516.28



506.80



467.35*



SD



21.27



36.30



41.60



38.99



N



10



10



10



10



%Diff



-



‑0.76



‑2.59



‑10.17



36 [a]



Mean



542.31



525.28



525.09



457.81*



SD



28.86



49.99



44.28



38.74



N



10



10



10



10



%Diff



-



‑3.14



‑3.18



‑15.58



43 [a]



Mean



555.10



542.65



523.81



469.06*



SD



26.78



45.01



40.11



37.97



N



10



10



10



10



%Diff



-



‑2.24



‑5.64



‑15.50



Absolute Weight Gain (g)



1 → 43 [a]



Mean



120.27



107.50



91.02



37.47*



SD



23.63



27.62



18.94



35.42



N



10



10



10



10



%Diff



.



‑10.62



‑24.32



‑68.84



[a] ‑ Anova & Dunnett: * = p < 0.05


 


Summary of Body Weights – Females









































































































































Sex: Female


Day(s) Relative to Start Date



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Body Weight (g)



1 [a]



Mean



274.70



274.70



276.36



275.11



SD



13.79



11.85



12.07



11.34



N



10



10



10



10



%Diff





0.00



0.60



0.15



8 [a]



Mean



285.13



278.35



283.35



267.83



SD



17.85



10.94



12.74



16.64



N



10



10



10



10



%Diff





‑2.38



‑0.62



‑6.07



15 [a]



Mean



294.75



289.55



295.36



264.79*



SD



21.99



12.86



15.88



22.67



N



10



10



10



10



%Diff





‑1.76



0.21



‑10.16



Absolute Weight Gain (g)



1 → 15 [a1]



Mean



20.05



14.85



19.01



‑10.32*



SD



11.09



5.12



7.87



17.60



N



10



10



10



10



%Diff



.



‑25.93



‑5.20



‑151.45



[a] ‑ Anova & Dunnett: * = p < 0.05


[a1] ‑ Anova & Dunnett(Rank): * = p < 0.05


 


Summary of Food Consumption – Males             








































































































































Sex: Male


Day(s) Relative to Start Date



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Food Con-sumption (g/animal/ day)



1 → 8 [a]



Mean



29.90



29.77



28.37*



20.43*



SD



0.64



1.30



0.27



1.37



N



10



10



10



10



%Diff



.



‑0.43



‑5.09



‑31.66



8 → 15 [a1]



Mean



31.17



29.94



29.25*



25.90*



SD



1.67



1.49



1.09



2.17



N



10



10



10



10



%Diff



.



‑3.94



‑6.15



‑16.91



29 → 36 [a]



Mean



32.23



30.52



28.70*



24.97*



SD



1.97



2.33



1.21



1.70



N



10



10



10



10



%Diff



.



‑5.30



‑10.96



‑22.53



36 → 43 [a]



Mean



32.38



28.28



28.17*



24.99*



SD



3.22



6.31



1.66



2.99



N



10



10



10



10



%Diff



.



‑12.64



‑12.97



‑22.80



[a] ‑ Anova & Dunnett(Rank): * = p < 0.05


[a1] ‑ Anova & Dunnett: * = p < 0.05


 


Summary of Food Consumption – Females         















































































Sex: Female


Day(s) Relative to Start Date



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Food Con-sumption (g/animal/ day)



1 → 8 [a]



Mean



21.49



20.14*



20.67



17.18*



SD



1.23



0.61



0.95



0.34



N



10



10



10



10



%Diff



.



‑6.28



‑3.78



‑20.03



 



8 → 15 [a]



Mean



20.94



19.80*



21.16



19.92



SD



0.86



0.39



1.19



1.96



N



10



10



10



10



%Diff



.



‑5.48



1.03



‑4.91



[a] ‑ Anova & Dunnett(Rank): * = p < 0.05


 


Summary of Oestrous Cycle Length Prior to Mating










































Sex: Female


Day(s) Relative to Start Date



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Mean


Cycle


(days)



1‑15 [a]



Mean



4.0



4.2



4.0



4.1



SD



0.0



0.2



0.1



0.1



N



10



10



10



10



[a] ‑ Anova & Dunnett(Rank)


 


Summary of Maternal Body Weights and Weight Change during Gestation Period           





























































































































































































































































Sex: Female


Day(s) relative to mating (Litter A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Body Weight (g)



0 [a]



Mean



300.07



289.77



299.99



286.19



SD



30.69



13.91



17.33



19.35



N



9



10



10



10



%Diff





‑3.43



‑0.02



‑4.62



7 [a1]



Mean



328.20



324.25



329.85



318.46



SD



30.22



13.48



17.71



18.41



N



9



10



10



10



%Diff



 



1.20



0.50



2.97



14 [a]



Mean



365.81



364.03



372.48



357.29



SD



31.57



23.21



20.89



20.47



N



9



10



10



10



%Diff





‑0.49



1.82



‑2.33



20 [a]



Mean



448.16



448.29



444.81



406.53



SD



35.57



26.23



28.99



28.96



N



9



10



10



10



%Diff





0.03



‑0.75



‑9.29



Absolute Weight Gain (g)



0 → 7



Mean



28.14



34.49



29.85



32.27



SD



6.12



4.52



6.12



6.36



N



9



10



10



10



%Diff



.



22.56



6.10



14.70



7 → 14



Mean



37.60



39.77



42.64



38.83



SD



5.05



12.23



7.43



9.68



N



9



10



10



10



%Diff



.



5.76



13.38



3.26



14 → 20



Mean



82.35



84.26



72.33



49.24



SD



8.63



16.57



20.20



21.72



N



9



10



10



10



%Diff



.



2.32



‑12.17



‑40.21



0 → 20



Mean



148.09



158.52



144.82



120.34



SD



9.42



15.96



19.70



30.68



N



9



10



10



10



%Diff



.



7.04



‑2.21



‑18.74



[a] ‑ Anova & Dunnett: * = p < 0.05           


[a1] ‑ Anova & Dunnett(Log)     


 


Summary of Food Consumption during Gestation Period             





























































































































































































































































Sex: Female


Day(s) relative to mating (Litter A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Total Food Consumption (g)



0 → 7 [a]



Mean



173.06



176.52



178.10



173.43



SD



9.81



6.91



11.31



13.75



N



9



10



10



10



%Diff



.



2.00



2.91



0.21



7 → 14 [a]



Mean



180.47



178.91



185.40



186.92



SD



18.76



12.19



26.73



15.53



N



9



10



10



10



%Diff



.



‑0.87



2.73



3.57



14 → 20 [a]



Mean



168.31



170.60



169.16



163.2



SD



10.64



12.30



15.17



14.75



N



9



10



10



10



%Diff



.



1.36



0.51



‑3.01



0 → 20 [a]



Mean



521.85



526.03



532.66



523.59



SD



34.79



25.36



41.66



37.63



N



9



10



10



10



%Diff



.



0.80



2.07



0.33



Food


Consumption


(g/animal/day)



0 → 7 [a1]



Mean



24.72



25.22



25.44



24.78



SD



1.40



0.99



1.62



1.96



N



9



10



10



10



%Diff



.



2.00



2.91



0.21



7 → 14 [a1]



Mean



25.78



25.56



26.49



26.70



SD



2.68



1.74



3.82



2.22



N



9



10



10



10



%Diff



.



‑0.87



2.73



3.57



14 → 20 [a1]



Mean



28.05



28.43



28.19



27.21



SD



1.77



2.05



2.53



2.46



N



9



10



10



10



%Diff



.



1.36



0.51



‑3.01



0 → 20 [a1]



Mean



26.09



26.30



26.63



26.18



SD



1.74



1.27



2.08



1.88



N



9



10



10



10



%Diff



.



0.80



2.07



0.33



[a] ‑ Anova & Dunnett(Log)


[a1] ‑ Anova & Dunnett


 


Summary of Maternal Body Weights and Weight Change during Lactation Period            



































































































































































































Sex: Female


Day(s) relative to littering (litter A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Body Weight (g)



0 [a]



Mean



347.22



337.55



345.13



316.12



SD



30.65



26.47



19.15



32.92



N



9



10



10



10



%Diff





‑2.78



‑0.60



‑8.96



4 [a]



Mean



357.84



352.79



361.73



320.96*



SD



28.67



28.14



25.56



28.94



N



9



10



10



10



%Diff





‑1.41



1.09



‑10.31



13 [a]



Mean



369.60



365.46



365.21



325.95*



SD



23.81



25.75



20.94



35.10



N



9



10



10



10



%Diff





‑1.12



‑1.19



‑11.81



Absolute Weight Gain (g)



0 → 4 [a]



Mean



10.62



15.24



16.60



4.83



SD



13.92



15.23



20.59



18.84



N



9



10



10



10



%Diff



.



43.43



56.28



‑54.50



4 → 13 [a1]



Mean



11.76



12.67



3.48



5.00



SD



13.92



11.65



9.22



27.79



N



9



10



10



10



%Diff



.



7.76



‑70.44



‑57.50



0 → 13 [a1]



Mean



22.38



27.91



20.08



9.83



SD



14.74



15.91



15.87



29.24



N



9



10



10



10



%Diff



.



24.69



‑10.29



‑56.08



[a] ‑ Anova & Dunnett: * = p < 0.05


[a1] ‑ Anova & Dunnett(Rank)


 


Summary of Food Consumption during Lactation Period              



































































































































































































Sex: Female


Day(s) relative to littering (litter A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Total Food Consumption (g)



0 → 4 [a]



Mean



168.39



166.98



151.32



118.41*



SD



56.92



33.22



16.95



21.48



N



9



10



10



10



%Diff



.



‑0.84



‑10.14



‑29.68



4 → 13 [a1]



Mean



468.99



519.25



485.59



397.70*



SD



57.73



54.08



57.84



70.37



N



9



10



10



10



%Diff



.



10.72



3.54



‑15.20



0 → 13 [a1]



Mean



637.38



686.23



636.91



516.11*



SD



105.56



74.81



59.28



76.91



N



9



10



10



10



%Diff



.



7.66



‑0.07



‑19.03



Food Consumption (g/animal/day)



0 → 4 [a]



Mean



42.10



41.74



37.83



29.60*



SD



14.23



8.31



4.24



5.37



N



9



10



10



10



%Diff



.



‑0.84



‑10.14



‑29.68



4 → 13 [a1]



Mean



52.11



57.69



53.95



44.19*



SD



6.41



6.01



6.43



7.82



N



9



10



10



10



%Diff



.



10.72



3.54



‑15.20



0 → 13 [a1]



Mean



49.03



52.79



48.99



39.70*



SD



8.12



5.75



4.56



5.92



N



9



10



10



10



%Diff



.



7.66



‑0.07



‑19.03



[a] ‑ Anova & Dunnett(Log): * = p < 0.05


[a1] ‑ Anova & Dunnett: * = p < 0.05


 


Summary of Fertility Data           





















































































































































































































































































































































Sex: Both


Day(s) Relative to Mating (Litter: A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Paired Males





N+ve



10



10



10



10



Paired Females





N+ve



10



10



10



10



Number of Males Mated





N+ve



10



10



10



10



 



%



100.0



100.0



100.0



100.0



 



ProA



10/10



10/10



10/10



10/10



Number of Females Mated



‑ [f]



N+ve



10



10



10



10



 



%



100.0



100.0



100.0



100.0



 



ProA



10/10



10/10



10/10



10/10



Pre‑coital Interval (f) (days)



‑ [a]



Mean



3.10



3.60



3.30



2.90



 



SD



2.28



1.65



2.31



2.85



 



N



10



10



10



10



Fertile Male?



‑ [f]



N+ve



9



10



10



10



 



%



90.0



100.0



100.0



100.0



 



ProA



9/10



10/10



10/10



10/10



Fertile Female?



‑ [f]



N+ve



9



10



10



10



 



%



90.0



100.0



100.0



100.0



 



ProA



9/10



10/10



10/10



10/10



Pregnant?



‑ [f]



N+ve



9



10



10



10



 





90.0



100.0



100.0



100.0



 



ProA



9/10



10/10



10/10



10/10



Littered



‑ [f]



N+ve



9



10



10



10



 



%



100.0



100.0



100.0



100.0



 



ProA



9/9



10/10



10/10



10/10



Gest. Length (days)



‑ [a]



Mean



22.1



22.4



22.4



22.5



 



SD



0.3



0.5



0.5



0.5



 



N



9



10



10



10



Male Mating Index



‑ [f]



%



100.0



100.0



100.0



100.0



 



ProA



10/10



10/10



10/10



10/10



Female Mating Index



‑ [f]



%



100.0



100.0



100.0



100.0



 



ProA



10/10



10/10



10/10



10/10



Male Impregnated Female



‑ [f]



%



90.0



100.0



100.0



100.0



 



ProA



9/10



10/10



10/10



10/10



Female Fertility Index



‑ [f]



%



90.0



100.0



100.0



100.0



 



ProA



9/10



10/10



10/10



10/10



Mated/Pregnant/Littered





N+ve



9



10



10



10



Mated/Not Pregnant





N+ve



1



0



0



0



Mated/Pregnant/Viable litter





N+ve



9



10



10



10



Mated/Pregnant/ No Viable litter





N+ve



0



0



0



0



[f] ‑ Chi‑Squared & Fisher's Exact


[a] ‑ Anova & Dunnett(Rank)    


 


Summary of Litter Data











































































































































































































































































































































































Sex: Female


Day(s) Relative to Littering (Litter: A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Total No of Pups Born



LD‑0 [a]



Mean



15.7



15.6



14.2



10.8*



N



9



10



10



10



Sum



141.0



156.0



142.0



108.0*



Dead/Canibalized on D0



LD‑0 [a1]



Sum



1



1



.



3



ILD – Live (M)



LD‑0 [a1]



Sum



67



76



81



52



ILD – Live (F) Females onD0



LD‑0 [a1]



Sum



73



79



61



53



ILD ‑ Live Pups on Day 0



LD‑0 [a]



Mean



15.6



15.5



14.2



10.5*



SD



1.0



2.5



2.3



4.1



N



9



10



10



10



Sum



140.0



155.0



142.0



105.0*



Live Pups Day 1



LD‑0 [a]



Mean



15.6



15.5



14.2



10.5*



N



9



10



10



10



Sum



140.0



155.0



142.0



105.0*



Dead/Cannibalized Pups D2‑4



LD 2‑4



Sum



1



.



.



.



Live Pups Day 4



LD‑4 [a]



Mean



15.44



15.50



14.20



10.50*



N



9



10



10



10



Sum



139.00



155.00



142.00



105.00*



No. of Pups Culled D4



4 [a1]



N



9



10



10



10



 



 



Sum



67



75



62



33*



No.of Live Pups Post Cull



LD‑04 [a]



Mean



8.0



8.0



8.0



7.2



SD



0



0



0



2



N



9



10



10



10



Sum



72



80



80



72



Dead/Cannibalized D2‑4



LD‑13



Sum



0



0



0



0



Live Pups Day 13



LD‑13 [a]



Mean



8.0



8.0



8.0



7.2



SD



0.0



0.0



0.0



1.9



N



9



10



10



10



Sum



72.0



80.0



80.0



72.0



Live Birth Index (%) D0



LD‑0 [f]



Mean



99.3



99.4



100.0



98.0



SD



2.1



2.0



0.0



6.3



N



9



10



10



10



Sex Ratio D0 (%)



LD‑0 [a1]



Mean



48.2



49.9



56.3



47.8



SD



11.0



14.4



17.8



17.2



N



9



10



10



10



% Survival Idx on LD4



LD‑4 [a]



Mean



99.3



100.0



100.0



100.0



SD



2.1



0.0



0.0



0.0



N



9



10



10



10



Mean No. of Implantation



‑ [a]



Mean



16.44



17.40



15.60



12.50*



SD



1.01



1.65



2.01



4.40



N



9



10



10



10



Sum



148.00



174.00



156.00



125.00*



% Post‑impt' Loss (%)



‑ [a]



Mean



5.3



11.1



9.1



16.9



SD



4.6



11.1



8.1



14.2



N



9



10



10



10



[a] ‑ Anova & Dunnett(Rank): * = p < 0.05


[a1] ‑ Anova & Dunnett: * = p < 0.05;


[f] ‑ Chi‑Squared & Fisher's Exact:


 


Summary of Litter Mean Pup Body Weights and Anogenital Distance
























































































































































































































































































































Sex: Female


Day(s) Relative to Littering (Litter: A)



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Mean Female Pup BW/L



0 [a]



Mean



6.237



6.496



6.808



6.567



SD



0.644



0.504



0.888



0.964



N



9



10



10



10



4 [a]



Mean



9.879



10.183



10.992



10.535



SD



0.919



1.041



1.207



1.763



N



9



10



10



10



13 [a]



Mean



29.665



31.101



31.914*



25.766



SD



2.418



1.563



1.302



4.484



N



9



10



10



10



Mean Male Pup BW/L



0 [a]



Mean



6.652



6.885



7.129



6.851



SD



0.636



0.675



0.975



0.931



N



9



10



10



10



4 [a1]



Mean



9.893



10.417



11.340



10.534



SD



0.936



1.376



1.169



1.773



N



9



10



10



10



13 [a1]



Mean



30.598



31.528



32.569



25.708*



SD



2.720



1.920



2.276



4.129



N



9



10



10



10



Mean Total Pup BW/L



0 [a2]



Mean



6.444



6.699



7.003



6.702



SD



0.622



0.628



0.876



0.924



N



9



10



10



10



4 [a1]



Mean



9.871



10.302



11.193



10.522



SD



0.916



1.217



1.174



1.765



N



9



10



10



10



13 [a]



Mean



30.132



31.315



32.229



25.746*



SD



2.469



1.591



1.686



4.297



N



9



10



10



10



Mean of pup AGR females



LD‑0 [a]



Mean



0.90



0.88



0.90



0.92



SD



0.03



0.00



0.03



0.06



N



9



10



10



10



Mean of pup AGR males



LD‑0 [a1]



Mean



1.95



1.94



1.94



1.96



SD



0.03



0.03



0.05



0.05



N



9



10



10



10



Mean of pup AGI females



LD‑0 [a]



Mean



0.9



0.9



0.9



0.9



SD



0.0



0.0



0.0



0.1



N



9



10



10



10



Mean of pup AGI males



LD‑0 [a]



Mean



1.9



1.9



1.9



2.0



SD



0.0



0.0



0.0



0.1



N



9



10



10



10



[a] ‑ Anova & Dunnett(Rank): * = p < 0.05


[a1] ‑ Anova & Dunnett: * = p < 0.05


[a2] ‑ Anova & Dunnett(Log)


 


Summary of Vaginal Smear Examination Prior to Necropsy         























































































































































































































































































Sex: Female


Day(s) Relative to Start Date



G1



G2



G3



G4



0 mg/kg/day



50 mg/kg/day



200 mg/kg/day



500 mg/kg/day



Stage of Estrus is E



43



N+ve



0



.



.



.



52



N+ve



0



.



0



.



53



N+ve



0



.



0



0



54



N+ve



0



0



0



.



55



N+ve



0



0



0



0



56



N+ve



.



0



0



.



60



N+ve



0



0



0



.



61



N+ve



.



.



.



0



Stage of Estrus is M



43



N+ve



0



.



.



.



52



N+ve



0



.



0



.



53



N+ve



0



.



0



0



54



N+ve



0



0



0



.



55



N+ve



0



0



0



0



56



N+ve



.



0



0



.



60



N+ve



0



0



0



.



61



N+ve



.



.



.



0



Stage of Estrus is D



43



N+ve



1



.



.



.



52



N+ve



1



.



1



.



53



N+ve



2



.



2



4



54



N+ve



2



6



3



.



55



N+ve



2



2



1



3



56



N+ve



.



1



2



.



60



N+ve



1



1



1



.



61



N+ve



.



.



.



1



Stage of Estrus is P



43



N+ve



0



.



.



.



52



N+ve



0



.



0



.



53



N+ve



0



.



0



2



54



N+ve



1



0



0



.



55



N+ve



0



0



0



0



56



N+ve



.



0



0



.



60



N+ve



0



0



0



.



61



N+ve



.



.



.



0



 

Applicant's summary and conclusion

Conclusions:
Considering the test item related adverse changes observed in body weight, body weight gains, food consumption, number of implantations, post-implantation loss, mean litter size, mean viable litter size, mean weight of pups organ weight and microscopic changes at 500 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Maternal/Reproduction/Developmental Toxicity for the test item 1,4-bis(2,3-epoxypropoxy)butane is determined to be 200 mg/kg bw/day under the test conditions and doses employed.
Executive summary:

To summarize, daily oral (gavage) administration of the test item “1,4-butanediol-bis(2,3-epoxypropoxy)” (also known as 1,4-butane diglycidylether, GRILONIT RV 1806, ERC #21) to Sprague-Dawley rats at the dose levels of 50, 200 and 500 mg/kg bw/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, during pregnancy and until 13 days after delivery (females) had no effects on general health, oestrous cycle length, pre-coital time, gestation length, mating and fertility parameters. There were no mortalities observed during the treatment at all the tested doses.


The transient clinical sign of slight salivation was observed from treatment Day 32 at 500 mg/kg bw/day and from Day 41 at 200 mg/kg bw/day soon after the dose administration.  The salivation was subsided within few minutes after dose administration which is common in gavage studies and hence, considered of no toxicologically significance. Lower mean body weight (males: 10.17 to 15.58%, females: up to 10.16%) and body weight gains were observed at 500 mg/kg bw/day in males and females, associated with decreased food consumption (males: 16.91 to 31.66%, females: up to 20.03%). The maternal gestation body weight (9.29%) and absolute weight gains (18.74 to 40.21%) were lower at 500 mg/kg bw/day, associated with decreased food consumption. The maternal lactation body weight (10.31 to 11.81%) and absolute weight gains (54.50 to 57.50%) were also lower at 500 mg/kg bw/day, associated with decreased food consumption.


At 500 mg/kg bw/day, the decrease in the mean litter size and mean viable litter size were observed on Lactation Days 0, 1 and 4, compared to the vehicle control group. The lower mean litter size at 500 mg/kg/day was due to the significantly lower number of implantations (24%) and higher post-implantation loss (16.9% vs 5.3% in vehicle control), when compared to vehicle control group. The mean body weight of male pups and total litter mean pups were lower on Day 13 at 500 mg/kg bw/day, compared to the vehicle control group.


The decrease in terminal fasting body weight at 500 mg/kg bw/day in adult males and females was considered as test item-related. Decreased thymus weight at 500 mg/kg bw/day in adult males and females were considered as stress-related changes. Decreased weights of prostate, Levator Ani/Bulbospongiosus Muscle at 500 mg/kg bw/day and seminal vesicles with coagulating glands at all dose levels were observed in adult males. These were considered as secondary change due to decrease in body weights at 500 mg/kg bw/day.


Grossly observed multiple white focus and multifocal thickening of non-glandular stomach at 500 mg/kg bw/day in adult males and females were considered as test item-related and microscopically correlated with ulcer and hyperplasia/hyperkeratosis, respectively. Multifocal red discoloration of non-glandular stomach at 500 mg/kg bw/day in adult males was considered as test item-related change without any histologic correlation.


Microscopically, stomach revealed ulcer, hyperplasia/hyperkeratosis, submucosal inflammation of non-glandular region and inflammation of pyloric region (glandular stomach) at 500 mg/kg bw/day in both sexes and were considered as test item-related adverse changes. At 200 mg/kg bw/day, the hyperplasia/hyperkeratosis of non-glandular mucosa observed in both sexes was considered as non-adverse effect of test item.


Considering the test item related adverse changes observed in body weight, body weight gains, food consumption, number of implantations, post-implantation loss, mean litter size, mean viable litter size, mean weight of pups organ weight and microscopic changes at 500 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for maternal and reproduction/developmental toxicity for the test item 1,4-bis(2,3-epoxypropoxy)butane is determined to be 200 mg/kg bw/day under the test conditions and doses employed. Effects seen on number of implantations, implantation losses, pup weights and pup organ weights were only seen at the high dose group correlating with significant maternal effects. No effects on fertility were observed.