Registration Dossier
Registration Dossier
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EC number: 604-617-1 | CAS number: 147993-66-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (propylene glycol), 15, 45 and 90 mg/kg bw/day in a key combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD No. 422). The NOAEL for reproductive effects of the parental generation was 45 mg/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 15 mg solid/kg bw/day.
Link to relevant study records
- Endpoint:
- three-generation reproductive toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- See attached read-across justification
- Reason / purpose for cross-reference:
- read-across source
- GLP compliance:
- yes (incl. QA statement)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related clinical observations for F0 males and females. Alopecia was noted with similar frequency in the control and treated groups.A common occurence in rodents maintained on ground feed is malocclusion. No parental females (F0) aborted or had physical or behavioral abnormalities during F1 gestation and lactation.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Survival was 100% for all groups of males and females at termination of the F0 generation animals except for the high-dose males where it was 97%. One male in this group (Animal No. C25201) was sacrificed at Week 10 in moribund condition due to an apparent fracture of the nasal bones.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Dose levels of 0.5% and 1.0% caused a reduction in body weight for the F0 males and the values were statistically significant at Weeks 9, 10, 18 and 19 for the 1.0% dose level and at Week 17 for the 0.5% dose level. No effect on body weight was seen for females at any dose level during the premating phase or during gestation. Although body weights for females during lactation were slightly lower than those of controls, the differences were not statistically significant.
There were no consistent significant differences in body weight gains for males. Body weight gains for 1.0% dose level females during Gestation Days 14 to 20 and 0 to 20 were significantly lower than those of controls . - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Although food consumptions for treated males were slightly lower than those of controls throughout the premating phase, these differences were significant only during Week 4 for the 0.5% dose level and during Weeks 4 and 5 for the 1.0% dose level. Food consumptions for 1.0% dose level females were lower than those of controls throughout lactation and statistically significant for all intervals except Days 10 through 14 and Days 17 through 21.
Compound consumption across groups was approximately proportional to the level of DSS administered in the test diets. As one would expect, it was decreased during the premating growth phase for both sexes(although to a greater extent in males ), remained low during gestation, and then increased appreciably during lactation. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There were no significant differences from the control group for male fertility indices during breeding for F1 litters. Fertility in males was defined by a female giving birth to a litter.
No significant differences from controls were observed for mating, fertility, or gestation indices, days required to mate, or the length of gestation. No parental females aborted or had physical or behavioral abnormalities during F1 gestation and lactation. - Dose descriptor:
- NOAEC
- Remarks:
- Generat: maternal/paternal toxicity
- Effect level:
- 0.1 other: %
- Based on:
- act. ingr.
- Remarks:
- in the diet (read-across test item)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- reproduction/offspring
- Effect level:
- 1 other: %
- Based on:
- act. ingr.
- Remarks:
- in the diet (read-across test item)
- Sex:
- male/female
- Basis for effect level:
- other: no effects up to highest concentration
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related clinical observations for F1 males and females. Alopecia was noted with similar frequency in the control and treated groups .
As in the F0 generation, there were several instances of malocclusion and/or related signs. When necessary, the incisors of affected animals were clipped to alleviate the condition.
No parental females (F1) aborted or had physical or behavioral abnormalities during F2 gestation and lactation. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- Survival was 100% for all groups of males and females at termination of the F1 generation animals.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights for F1 males and females at the 1.0% dose levels were significantly lower than those of controls throughout the premating phase, excluding Week 2 for females, and throughout the treatment period for males. Body weights for F1 males and females in the 0.5%. dose group were also low (although not statistically significant) during the entire premating phase.
Gestation body weights tor females were significantly lower than those of controls at the1.0% dose level throughout gestation. Body weights during lactation were significantly lower on days 0, 7, and 14 at the 1.0% dose level; body weights on Lactation Day 21 at the 1.0% dose level were also lower than those of controls, although this difference was not statistically significant.
Body weight gains were significantly lower than those of controls during Weeks 5 and 6 for males and during Weeks 3, 5, and 8 for females at the 1.0% dose level, and during Weeks 5 and 10 for females at the 0.5% dose level. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food consumptions were significantly lower than those of controls throughout the premating phase· (except f or Week 3) for males at the 1.0% dose level, and during Weeks 6 through 9 for males at the 0.5% dose level. Female food consumptions were significantly lower than those of controls during Weeks 7 and 8 at both the 0.5% and 1.0% dose levels.
Food consumptions during gestation were significantly greater than those of control only during Days 0 through 4 at the 0.1% dose level. Food consumptions during lactation were significantly lower during Days 7 through 10 at the 0.5% dose level, and during Days 4 through 7, 7 through 10, and 14 through 17 at the 1.0% dose level. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related macroscopic observations in any animals examined in the study (F0, F1 and F2 adults and F3 weanlings).
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There were no significant differences from the control group for male fertility indices during breeding for F2 litters .
No significant differences from controls were observed for mating, fertility, or gestation indices, days required to mate, or the length of gestation. No parental females aborted or had physical or behavioral abnormalities during F2 gestation and lactation. - Dose descriptor:
- NOAEC
- Remarks:
- maternal/paternal toxicity
- Effect level:
- 0.1 other: %
- Based on:
- act. ingr.
- Remarks:
- diet (read-across test-item)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- reproduction/offspring
- Effect level:
- 1 other: %
- Based on:
- act. ingr.
- Remarks:
- diet (read-across test item)
- Sex:
- male/female
- Basis for effect level:
- other: no effects up to highest concentration
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio. No pups from F1 litters had external abnormalities.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Male and female pup weights on days 4, 7, 14, and 21 and pup weight gains from Days 0 through 4, 4 through 7, 7 through 14, and 14 through 21 at the 1.0% dose level were significantly lower than those of controls. Male and female pup weights on day 21 and weight gains from Days 7 through 14 (females only), and from days 14 through 21 (males and females) for the 0.5% dose level were significantly lower than those of controls.
- Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- Pups were allowed to nurse for 21 days before weaning. Food consumption after weaning is described under the parental generation.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No pups from F1 litters had external abnormalities.
- Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- 0.1 other: %
- Based on:
- act. ingr.
- Remarks:
- diet read-across test-item)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio.
Alopecia, tremors, and rough hair coat were recorded on day 21 for three pups (two males and one female) from one F2 litter at the 0.5% dose level. - Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Beginning on day 7, male and female pup weights and pup weight gains at the 0.5% and 1.0% dose levels were consistently lower than those of controls; the differences were statistically significant for all values except day 7 female pup weight, male and female pup weight gains for the 0.5% dose level. On Day 4 of lactation no milk was observed in the abdomen of three pups from two litters in the control group, seven pups from five litters in the 0.1% dose group, 18 pups from 10 litters in the 0.5% dose group, and 10 pups from five litters in the 1.0% dose group.
- Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- Pups were allowed to nurse for 21 days before weaning. Food consumption after weaning is described under the parental generation.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Alopecia, tremors, and rough hair coat were recorded on day 21 for three pups (two males and one female) from one litter at the 0.5% dose level.
- Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEC
- Generation:
- F2
- Effect level:
- 0.1 other: %
- Based on:
- act. ingr.
- Remarks:
- diet (read-across test item)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Reproductive effects observed:
- no
- Lowest effective dose / conc.:
- 1 other: %
- Conclusions:
- Read-across test item DSS administered in the diet to three successive generations of rats at levels of 0.5% and 1.0% caused a reduction in body weights for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and the test substance had no adverse effects at levels on the reproductive function of either sex in any generation up to 1% in the diet.
Based on the results of this study, when DSS was administered in the diet to three succesive generations of rats, the no-observable-effect level (NOEL) for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0% DSS. - Executive summary:
Read-across test item docusate sodium was administered in the diet to three successive generations at levels of 0.1%, 0.5% and 1.0% in the diets of 30 males and 30 females per group, dosed for 10 and 2 weeks respectively. The dose levels of 0.5% and 1.0% caused a reduction in body weights for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and the test substance had no adverse effects at levels on the reproductive function of either sex in any generation up to 1.0% in the diet. There were no other effects on parental or reproductive parameters.
Based on the results of this study, when DSS was administered in the diet to three succesive generations of rats, the no-observable-effect level (NOEL) for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0% DSS.
- Endpoint:
- reproductive toxicity, other
- Remarks:
- Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 April 2021 (study plan) – 03 November 2022 (final report)
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:WI (Wistar)
- Details on species / strain selection:
- Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 12-13 weeks old (females/males) at start of the experiment
- Weight at study initiation: Males: 409-512 g, females: 244-298 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: no information
- Housing: Rodents were group-housed, up to 2 animals of the same sex and group/cage in type II, III and/or IV polycarbonate cages. During the mating and gestation, delivery, lactation period, they were paired or individually housed (with pups), respectively. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201208, Expiry date: 08 December 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200824 / 05072201021, Expiry date: 24 August 2023 / 21 October 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch numbers: A/123/D and A/123/E) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch numbers: 713 70882 / 187 76795, Expiry dates: 30 April 2021 / 31 August 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: 19 days
DETAILS OF FOOD AND WATER QUALITY:
The food was routinely analysed and considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The quality control analysis of the water was performed once every three months and microbiological assessment was performed monthly, by National Institute of State Public Health and Medical Officer Service. The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 – 25.9°C (target: 22 ± 3°C)
- Humidity (%): 30 - 61% (target: 30 - 70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
IN-LIFE DATES: From: 13 April 2021 (first vaginal smear sampling) To: 21 June 2021 (last necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- propylene glycol
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 6 days prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/127-901AN). Based on those results, the test item formulation in the 1 and 200 mg/mL concentration range were stable for at least 7 days when stored at room temperature.
The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with ultrasonication and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied to aid dissolution.
VEHICLE: Propylene glycol
- Concentration in vehicle: 0, 3, 9 and 18 mg/mL for the dose levels group of 0, 15, 45 and 90 mg/kg bw/day, respectively.
- Amount of vehicle (if gavage): A constant volume of 5 mL/kg bw was administered to all animals
- Lot/batch no.: 1920944 - Details on mating procedure:
- - M/F ratio per cage: 1:1 mating
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy: A vaginal smear was prepared daily during the mating period and stained with 1% (w/v) aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Sample collection was performed on three occasions (during the first and last weeks and approximately midway during the treatment period). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Pharmacy Department.
On each sampling occasion, top, middle and bottom duplicate samples was taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which can be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
Analysis of control (vehicle) and test item formulations for concentration and homogeneity was performed at the Test Site for analytical work was using a validated analytical method (20/127-901AN). Representative samples of control (vehicle) and test item formulations were analysed at three times during the study (during the first and last weeks and once approximately midway during the treatment period).
Formulation samples were kept at room temperature until shipment. Samples to be analysed was shipped to the Test Site as soon as practical after collection for concentration measurements.
Analysis of the formulations for concentration and homogeneity of test item was performed using a validated analytical HPLC-MS method (High Performance Liquid chromatography–mass spectrometry) at the Test Site (Study code for method validation: 20/127-901AN).
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the RSD% (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 92.1% and 105.9 % of the nominal concentrations.
All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10%.
Formulations were considered to be adequately stable under the study conditions. - Duration of treatment / exposure:
- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. - Frequency of treatment:
- daily
- Dose / conc.:
- 15 mg/kg bw/day (actual dose received)
- Remarks:
- low dose
- Dose / conc.:
- 45 mg/kg bw/day (actual dose received)
- Remarks:
- mid dose
- Dose / conc.:
- 90 mg/kg bw/day (actual dose received)
- Remarks:
- high dose
- No. of animals per sex per dose:
- 12 animals/sex/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The oral route was selected as it is one of the possible routes of human exposure. The way of test item and vehicle control item administration was in compliance with the relevant OECD No. 422 guideline.
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/127-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study all the 1000 mg/kg bw/day dose group animals died or were euthanized, 2/4 female and 2/4 males in the 600 mg/kg bw/day dose group died or were euthanized: 600 mg/kg bw/day group animals were treated two days, 300 mg/kg bw/day group animals were treated eight days, then remained untreated to get information on the recovery. 1/4 male and 4/4 females died in the 300 mg/kg bw/day dose group. No animals died in the 100 mg/kg bw/day group.
There were no clinical symptoms or severe body weight decrease seen at 100 mg/kg/day. A range of symptoms (hunched back, piloerection, decreased activity, faeces liquid / soft, noisy respiration, red discharge around nose, wasted and general poor condition) were seen in Mid dose, High dose and High dose 2 animals indicating adverse effects; with no treatment effects in the Low groups.
The Low dose group had reduced growth or weight loss in the first week, with a near normal growth rate thereafter. The Mid to High dose group survivors lost significant weight in the treatment phase, and continued to have weight loss even after treatment was halted.
The Low dose group had minor haematological differences of equivocal relationship with treatment. Low dose males had no substantial effects in clinical chemistry; low dose females had changes in serum enzymes suggestive of hepatic effects, but not indicative of a severe effect. Other group data for clinical pathology was either at euthanasia or after a period without test item treatment, hence are not useful for dose setting, although no pattern was seen to suggest any specific toxic mechanism.
Test item-related bilateral enlargement of the adrenal glands (indicative of stress) and all lobes of liver was observed in High dose 2 male. Multifocal red discolouration of the glandular stomach and multifocal thickness of non-glandular stomach (indicative of gastric irritation) was observed in two Mid dose males, one High dose 2 male and one High dose 2 female. Mild to moderate periportal hepatocellular vacuolation was seen in Low dose females; it is considered as non-adverse in the context of this study, but it may possibly progress with longer exposure. Treatment-related effects were observed in the adrenal gland weights in Mid and High dose 2 males and liver weights in Low and High dose 2 females. The Low dose males had no evident changes in organ weight.
The dose of 90 mg/kg/day is selected for the High dose. Lower doses were spaced with a factor of approximately 3.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
Any unused, spare animals were moved back to the stock colony after they were not needed for the study (after of treatment).
- Fasting period before blood sampling for clinical biochemistry: yes, overnight period of food deprivation, in case of females this happened after the litter had been necropsied - Positive control:
- Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT).
Cyclophosphamide Dose Level: 60 mg/kg bw - Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General (routine) clinical observations were made once a day*, during the pre-treatment and treatment period in the afternoon (pm). *Note: No general clinical observations were made on the day of necropsy. Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). Any animal (including also all premature decedents) which showed clinical signs considered severe was sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am), before treatment) and before necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and 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), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.
BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, on Day 0 (randomisation and the first day of exposure), and afterwards weekly, and at termination.
Parent females were weighed on the first day of dosing, then once a week until mating.
On Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0, 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD3, GD10 and GD17 as well as PPD10 were additional measurements as aid for the calculation of accurate treatment volumes, these data was evaluated statistically.
FOOD CONSUMPTION AND COMPOUND INTAKE (no feeding study):
Animal food consumption was determined at randomisation by weighing the non-consumed diet with a precision of 1 g at least weekly and on Day 0. Food consumption was measured during mating. Food consumption was measured on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20 and on PPD0, 4, 7, 10 and 13 during the lactation period).
Main daily food consumption was calculated for each interval.
WATER CONSUMPTION AND COMPOUND INTAKE (no drinking water study): No
No water consumption was measured in the study. - Oestrous cyclicity (parental animals):
- Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments started. Any females that fail to show a 4-5 days cycles were not included in the study. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating (during the pre-mating and mating periods).
Additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. - Sperm parameters (parental animals):
- For the adult animals, detailed histological examination was performed on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males) of all animals of the Control and High dose groups, and of all males that failed to sire. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.
On PND 4, litters were culled to yield 5 males and 5 females per litter (or as nearly as possible). Pups to be culled within each litter were selected random. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0), and on PND4, PND7 and PND13, with accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) were also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.
On PND 4, litters were culled to yield 5 males and 5 females per litter (or as nearly as possible). Pups to be culled within each litter were selected random. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). The anogenital distance was normalized to a measure of pup size (the cube root of body weight) for statistical analysis.
Number of nipples/areolae in male pups was recorded on PND13.
One male and one female pup per litter were previously selected for culling for blood sampling on PND4.
All pups were necropsied on PND13.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs.
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not assessed
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not assessed - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals one day after the last treatment
- Maternal animals: All surviving animals one day after the last treatment
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.
HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all surviving adult animals were determined:
•With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
•With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all surviving animals:.
Gross findings
Adrenals
Animal identification (Fixation and preservation only.)
Aorta (Aorta thoracic and abdominal.)
Brain (7 section according to the NTP recommendations)
Epididymis
Eye with the optic nerve (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections.)
Oesophagus
Femur with marrow
Heart (Section including both ventricles and atria, septum with papillary muscle.)
Kidney
Large intestine (Caecum, colon and rectum.)
Extraorbital lachrymal gland
Harderian gland
Liver (Liver, 3 lobes, left lateral, right medial, caudate.)
Lungs with bronchi (Lungs of euthanized animals was infused with formalin; 3 lobes, left, right cranial, right caudal.)
Lymph node (Mandibular and mesenteric.)
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine (Duodenum, ileum and jejunum with Peyer’s patches.)
Spinal cord (Transverse sections, 3 levels – cervical, thoracic and lumbar.)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix.)
Vagina
Additionally, thyroid glands from all adult animals and one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
•on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group), - number of animals was notified by a Memo
• any animals found dead or euthanized pre-terminally during the study in all groups,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males that failed to sire, total intrauterine mortality animals and all females that failed to deliver healthy pups (this was notified by Memo).
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow, and blood smears if examined).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
Additional histopathology examination on the liver of all animals (Control, Low, Mid and High dose) and on the prostate and seminal vesicle on all the males (Control, Low, Mid and High dose) was performed because it was deemed necessary by the Pathologist and the Study Director. - Postmortem examinations (offspring):
- SACRIFICE
- Those litters where there were more than 10 pups were culled on Post-natal Day (PND) 4. All selected F1 offspring were terminated on Post-natal Day 13 (F1 offspring selected for blood sampling were terminated on PND4 due to technical reason). In order to allow for overnight fasting of dams prior necropsy on PPD14, offspring was euthanized on PPD/PND13, and the dams on PPD/PND14.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities. All pups were necropsied on PND13.
GROSS NECROPSY
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs. Presence of nipples/areolae in the PND13 male pups was also recorded.
HISTOPATHOLOGY / ORGAN WEIGTHS
Additionally, thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.
No histopathological examination was performed on pups (F1 generation).
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. - Statistics:
- see "Any other information on materials and method incl. tables"
- Reproductive indices:
- Formulas for Calculation of Mating and Fertility Indices
Male Mating Index = (Number of males with confirmed mating / Total Number of males cohabited) x 100: Measure of male’s ability to mate
Female Mating Index = (Number of sperm-positive females / Total Number of females cohabited) x 100: Measure of female’s ability to mate
Male Fertility Index = (Number of males impregnating a female / Total Number of males cohabited) x 100: Measure of male’s ability to produce sperm that can fertilise eggs
Female Fertility Index = (Number of pregnant females / Number of sperm-positive females) x 100: Measure of female’s ability to become pregnant
Gestation Index = (Number of females with live born pups / Number of pregnant females) x 100: Measure of pregnancy that provides at least one live pup - Offspring viability indices:
- Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices
Survival Index % = (Number of live pups (at designated time) / Number of pups born) x 100
Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born
Pre-implantation mortality % = [(Number of Corpora lutea – Number of Implantations) / Number of Corpora lutea] x 100
Intrauterine mortality % = [(Number of Implantations – Number of liveborns) / Number of Implantations] x 100
Total mortality % = [(Number of implantations – Number of viable pups (PND0/4/13)) / Number of Implantations] x 100
Sex ratio % (females) =[Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)] x 100 - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item related clinical signs were observed in the study.
Liquid faeces were recorded during the lactation period (Day 52) for one Low dose female (#2503) and on Day 51 for a Mid dose female (#3505).
Red liquid in the vulva was observed for one High dose female (#4509) on Day 33 and 34. Piloerection was observed for this animal on Day 16.
Noisy respiration was observed for one Control female (#1503) on Day 6 and 7.
Hunched back and piloerection were observed for one Control female (#1510) form Day 17 to Day 20.
These findings were all considered as being incidental, not related to the test item administration. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- No test item related mortality was observed in the study.
One Control male (#1009) was found dead on Day 28. No clinical sings were recorded prior to death. The cause of death could not be determined.
One High dose male (#4004) was preterminally euthanised on Day 14. Hunched back, piloerection, and wasted were observed prior to euthanasia. The cause of the death was the poor clinical condition, which was probably treatment related, but no cause of the clinical effects was evident from the macroscopic or histological evaluation. One Low dose female (#2510) was preterminally euthanised on PND0 because of prolapsed vagina, considered to be unrelated to treatment. Moderate paleness (whole body) and piloerection were recorded prior to death. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item related adverse effect was observed on body weight gain parameters in High dose (90 mg/kg bw/day) males and females and Mid dose females (45 mg/kg bw/day). No test item related adverse effect on body weight or body weight gain was detected in Mid dose (45 mg/kg bw/day) male or Low dose (15 mg/kg bw/day) animals (males or females).
In the first week, High dose males lost 8.5% of their bodyweight, with weight gain thereafter. This High dose body weight loss, in the first week, was considered as a test item related effect.
No effect on body weight parameters was seen in Mid and Low dose males.
In case of females, the observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period in all dose groups. The Mid and High dose females showed reduced body weight / body weight gain at the end of the gestation period (body weight was lower by 7.8% (p<0.05) and 16.7% (p<0.01), body weight gain by 21.8% (p<0.01) and 42.2% (p<0.01)).
In case of High dose females, statistically significantly lower body weight parameters were recorded by the end of the lactation period (by 19.5% (p<0.01) lower body weight and by 87.4% (p<0.01) lower body weight gain), with virtually no weight gain at all during the lactation period. The body weight gain from Day 0 to PPD 13 was statistically significantly lower for Mid dose and High dose females than Control (by 14.8% (p<0.05) and 70.6% (p<0.01)). Body weight data from PND 7 in the High dose were outside of the historical control range. These body weight effects were considered as being a test item related adverse effect.
No effect on body weight parameters was seen in Low dose females. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Test item related adverse effects were observed on food consumption in High dose males and females (90 mg/kg bw/day) and Mid dose females (45 mg/kg bw/day) during the treatment period; Mid and Low dose males and Low dose females were not affected.
Statistically significantly decreased (p<0.01) food consumption was observed in the High dose male group (90 mg/kg bw/day) at an average of ~30% below control for the 4-week period. The other groups (Mid and Low) were unaffected.
Statistically significantly decreased food consumption was observed in the Mid (p<0.05) and High dose (p<0.05 and p<0.01) female groups during gestation and lactation periods. Premating the female food intakes were not significantly different to control (all ~11-15g/day); during gestation although there were statistical differences, all groups were within about 15% of the controls; during lactation normal control food intake goes up dramatically (from about 35g/day initially, up to ~70g/day by Days 10-13) but the High dose group had no normal increased food intake and remained at the pre-lactation range of generally <20g/day, the Mid dose group were generally within 15-20% of the control values, which is a relatively slight effect. Together with the body weight data, the observed values were considered as a test item related adverse effect, but at the High dose the very low food intake could not support a normal high-energy-requirement for the level of lactation needed for pup growth. - Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- There were statistically significantly changes in the male dosed groups sporadically, but all the data were within the historical control range therefore they were not considered as test item related effects. There was statistically significantly decreased reticulocyte relative (%) and mean cell volume observed in the High dose female group and the values were outside of the historical control range, hence it was considered as test item related effect.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- There were test item related effects observed in the High dose male and female groups on clinical chemistry parameters. There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration in the Low dose group for both sexes.
Statistically significantly decreased cholesterol concentration was observed in Mid dose male and High dose males and females (p<0.01), the data were outside of the historical control range. But this fact was most probably related to the significantly reduced food consumption of animals during the gestation and lactation periods, not considered as being a direct adverse effect.
Statistically significantly decreased total protein (p<0.01) was measured in the High dose male group (possibly related to food intake) but the data were within the historical control range.
Statistically significantly increased A/G ratio (p<0.05) was measured in the High dose male group, the data were outside of the historical control range, hence it was considered as test item related effect but probably related to food intake. There was statistically significantly increased (p<0.01) bile acid concentration observed in the High dose male group, the data were outside of the historical control range, therefore it was considered as a possible test item related effect. Statistically significantly increased chloride concentration (p<0.01) and decreased phosphorus concentration (p<0.05) was observed in the High dose female group, but the data were within the historical control range.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the Mid dose males (p<0.01) and High dose males (p<0.01) and females (p<0.01) when compared to control animals. In case of High dose females, the observed values were outside of the historical control range. Furthermore, increased Alkaline phosphate (ALKP) activity was recorded in the Mid dose males and females (p<0.01) and High dose males and females (p<0.01), the observed female mean values were within of the historical control range, the High dose male value was outside of the historical control range.
Statistically significantly increased urea concentration (p<0.01) was observed in the Mid dose female groups, which was outside of the historical control range but without dose response it was not considered as test item related effect. Statistically significantly increased potassium and calcium concentration (p<0.05) and was observed in the Mid dose female groups, which were within the historical control range.
Observed values of alanine aminotransferase (ALT/GPT), A/G (albumin/globulin) ratio and bile acid concentration in case of the High dose males were considered as test item related but none were of a magnitude to be considered clearly adverse. These changes were probably related to the low food intakes and/or the histopathology findings in the liver. - Endocrine findings:
- no effects observed
- Description (incidence and severity):
- In the parameters measured in adults and pups, there were no effects on thyroids or other endocrine systems.
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urinary blood (p<0.01) was observed in the High dose male group but in the absence of any histopathological kidney changes are not considered as a clear adverse effect of the test item. Statistically significantly decreased specific gravity (p<0.01) and protein level (p<0.05) was observed in the High dose female group. The specific gravity data were outside of the historical control range. Statistically significantly increased urine crystals (p<0.01) were observed in the High dose female group. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength. There was statistically significantly increased (p<0.05) landing splay (Hind paws) observed in the High dose male group, but the data were within the historical control range therefore these changes did not consider as test item related effects. There was statistically significantly increased (p<0.05) landing splay (Fore paws) observed in the Low and Mid dose male groups.
All dose groups of males and females had comparable locomotor activity to the Control. There was statistically significantly increased LMA distance (10-15 min) was observed in the Low dose male group without dose response. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data. - Immunological findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- -NON-PREGNANT FEMALES / Parental Generation
Microscopically in the #4505 female ovarian atrophy and dilatation of uterine horns were seen and considered to be incidental, in the male pair (#4005) of this female, minimal multifocal hepatocellular vacuolation, and minimal decreased secretion in the prostate and seminal vesicle were present (similarly to the terminal animals) which were considered as test item-related. Other findings in these two animals were considered procedure related, incidental or a common background.
In the #2512 female no microscopic findings were noted, in the male pair (#2012) of this female sinusoidal erythrocytosis was present in the mandibular lymph nodes, as procedure related (due to sublingual blood sampling).
-FOUND DEAD ANIMAL / Parental Generation
Microscopic Findings: Microscopically congestion/haemorrhage in the lungs and thymus; these were considered as agonal, or incidental.
-PRE-TERMINAL EUTHANASIA / Parental Generation
Microscopic Findings: No test item-related changes were observed microscopically for the Low dose female. In case of the High dose male the mandibular lymph nodes were dark red, the thymus was small, on the glandular gastric mucosa white and red foci were present at necropsy, microscopically and were considered as not test item related. The cause of termination of the male was considered to be treatment related clinical signs, but there was no evidence of pathological changes that could indicate the aetiology of the adverse clinical signs.
-TERMINAL EUTHANASIA / Parental Generation
Microscopic Findings: Test item-related multifocal/diffuse (mainly multifocal) hepatocellular vacuolation was detected in 10/11 High dose, in 4/12 Mid dose males and in 7/7 High dose and in 7/12 Mid dose females with minimal to marked severity (typically minimal/mild severity in males and moderate in the females), correlated with organ weight changes and necropsy findings.
In High dose males, in the prostate decreased secretion was seen in 7/11 cases, in the Mid dose in 2/12 cases with minimal/mild severity. Decreased secretion was detected in the seminal vesicles in 4/11 High dose males (correlated with organ weight changes) and were considered as test item related. Decreased secretion was detected in the seminal vesicles in 1/9 Low dose male as well and was considered as incidental. Similar findings in secondary sex organs are common when animals have a significant body weight loss in the first week of treatment.
In the kidney of all examined High dose males, minimal multifocal eosinophilic droplets/globules were seen in the renal tubules and were considered as test item related. These hyaline droplets are most likely to be Alpha 2u globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only; in the context of human safety evaluation, this rat-specific change is considered to be non-adverse.
All other findings were considered as incidental or background and not to represent adverse effects of the test item. - Histopathological findings: neoplastic:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- -Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00-4.04 days was observed in the different groups) before starting the treatment period.
-Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.08, 4.00, 4.02 and 4.11 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in 1 Mid dose female and 2 High dose females were affected (#3510, #4506 and #4509), but as it did not affect mating or pregnancy, this fact was considered as being an occasional finding, not being a test item related effect.
Prolonged dioestrus was not noted.
Pseudo-pregnancy was noted for one Mid (#3509) dose female, but this frequency was in line with the normal, expected range. - Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- There were test item related adverse effects for the High dose group with regard to reproductive ability or gestation indices. The mating index was 100% in all groups (males and females). The fertility index was 100%, 92%, 100% and 91% in the Control, Low, Mid and High dose groups for males and 100%, 92%, 100% and 92% in the Control, Low, Mid and High dose females. The gestation index was 100%, 100%, 100% and 55% in the Control, Low, Mid and High dose females.
Eleven females in the High dose group were pregnant. However, one female had no live born pups (#4502) and four pregnant females (#4507, #4509, #4510 and #4512) had no delivery. Corpora lutea and implantations were observed for these 4 females but 100% of the embryos were resorbed. It was considered as an adverse test item related effect, the number of females with complete resorption of embryos was well outside the historical control range.
There were statistically significantly decreased (p<0.05 and p<0.01) number of corpora lutea and pre-implantation loss in the Mid and High dose groups which were considered as test item related adverse effect.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation). The mean duration of mating was 2.58, 2.33, 2.33 and 2.25 days in the Control, Low, Mid and High dose groups, respectively.
There was effect of treatment noted during the gestation period, parturition and post-partum period in the Mid and High dose groups.
The mean duration of pregnancy was statistically significantly increased in the High dose group, the data were outside of the historical control range.
As far as it could be observed during the study, the parturition was normal for all animals, except one Low dose female (#2510), one Mid dose female (#3511) and two High dose females (#4502 and #4504).
The number of implantation sites was statistically significantly lower (p<0.01) in the High dose group compared to the Control, although the food intake was normal up to the time of implantation (~ GD7).
There were statistically significant differences that could be ascribed as treatment related on pre-natal, post-natal or total mortality values (litter mean and %) in the Mid and High dose groups compared to the Control. For High dose group the post-natal mortality and total mortality data were 100% which is far higher than historical control groups. The total intrauterine mortality value in the Mid dose group was outside of the historical control group range. For the High dose, the very low food intake by the dams would have precluded an adequate milk production, low growth and death of pups would be expected as secondary to the food intake effect. In the Mid dose a statistically lower food intake by the dams may have influenced the pup growth and mortality data. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- reproductive effects
- Effect level:
- 45 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- reproductive performance
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- Effect level:
- 15 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups.
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- There were test item effects on mortality or survival of the pups (F1 generation) in the Mid and High dose groups, associated with low food intake by the dams during lactation.
The mean number of viable pups born were statistically significantly decreased in the Low (p<0.05) and High dose groups (p<0.01). Pre-natal mortality for the Mid (p<0.05) and High (p<0.01) dose groups was statistically significantly increased compared to the Control. The Mid and High dose groups values were outside of the historical control range.
Total mortality in the High dose groups was outside of the historical control data and clearly an adverse effect (probably related to the very low food intake that was insufficient for adequate lactation). A similar trend in the Mid dose was statistically significantly increased compared to the control but was generally similar to the Low dose group where there were no statistical differences to control.
Evidence of suckling was recorded for all live born pups in the study except in case of two High dose dams (#4503 and #4506), where the pups were born alive but shortly after the end of the delivery were seen to have died (which was clearly not related to low milk production subsequent to low food intake but was considered to be an adverse effect of treatment). - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- There were test item related statistical differences in the offspring body weights or weight gains in the Mid dose group when compared to the controls, in the High dose group, the limited available data also indicates a weight growth effect. The measured values were within the historical control range for the Mid dose throughout the development phase.
Because of the lower maternal food consumption by 15-20% in the Mid dose, less milk and lower growth of the pups was observed, therefore this effect is considered as secondary to maternal effect. In the High dose, the 100% mortality prevents a proper assessment of pup growth. - Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- No test item effect was observed on anogenital distance during the study.
There were statistically significantly decreased anogenital distance values observed in the Mid and High dose male groups when compared to the Control, but all the data were within the historical control range and when adjusted for the cube root of body weight, therefore these effects were not considered as test item related . - Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- No test item effect was observed on nipple retention during the study. There was no nipples/areolae presence seen in any of the male pups on PND13.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No test item-related changes were observed in macroscopically examined pups.
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- pups' (F1 generation) development and survival
- Effect level:
- 15 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- viability
- mortality
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 45 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- The NOAEL for reproductive effects of the parental generation: 45 mg/kg bw/day
(based on marked adverse findings on pregnancy seen only in the High dose group, whereas statistical differences in the Mid dose were consistent with historical control ranges).
The NOAEL for pups’ (F1 generation) development and survival: 15 mg/kg bw/day
(based on the death of all High dose pups and based on Mid dose group statistically significant mortality data). - Executive summary:
The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of the test item Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (propylene glycol).
The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Post-natal Day (PND) 13.
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on the results of the DRF study, 90 mg/kg bw/day was selected as the High dose for this study. Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT).
Experimental design:
Group Number
Group designation
Dose level (mg/kg bw/day)
Concentration (mg/mL)
Dose volume (mL/kg bw)
Animal numbers
Male
Female
1
Control
0
0
5
1001-1012
1501-1512
2
Low Dose
15
3
2001-2012
2501-2512
3
Mid Dose
45
9
3001-3012
3501-3512
4
High Dose
90
18
4001-4012
4501-4512
Gr. No.
Group Designation
Cyclophosphamide Dose Level
(mg/kg bw)Cyclophosphamide Conc. (mg/mL)
Dose volume
(mL/kg bw)
Animal Numbers
Male
Female
5
Positive control MNT
60
12
5
5
5
Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis).
Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13.
At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND (Post-natal Day) 13 pups and parental males were also determined.
For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, the retained reproductive organs of all Control and High dose animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved.
RESULTS
In summary, under the conditions of this study the daily administration of Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts by oral gavage to Wistar rats at dose levels of 15, 45 or 90 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or clinical signs in the majority of animals. One High dose male had clinical signs of weakness and general poor condition, it was terminated early, but there was no pathology found that could indicate the aetiology of the test item effects; however the early termination is ascribed to treatment.
Test item related adverse effect was observed on body weight, body weight gain parameters and food consumption in High dose (90 mg/kg bw/day) males and females. Test item related adverse effect was observed on body weight, body weight gain parameters and food consumption in Mid dose (45 mg/kg bw/day) females. In males, the High dose animals lost ~8.5% body weight in the first week of the study but gained weight thereafter; lower food intake in the High dose female group during lactation would have resulted in lower milk production (associated with loss of all pups in this group).
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in test item treated groups when compared to control.
Statistically significant haematological changes comprised decreased reticulocytes (%) in the High dose female group with the value was outside of the historical control range, which was considered a test item related effect. Statistically significant clinical chemistry changes included increased alanine aminotransferase, ALT/GPT ratio and bile acid concentration in case of the High dose males were considered as test item related but none were of a magnitude to be considered clearly adverse. These changes were probably related to the low food intakes and/or the histopathology findings in the liver. No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Eleven females in the High dose group were pregnant. However, one female had no live born pups and four pregnant females had no delivery (100% of the embryos was resorbed). High dose pups who were live born were dead in three days after birth the latest. It was considered as test item related adverse effect.
There were severe test item related effects on pup mortality or survival of the pups (F1 generation) in the High dose groups; slight but similar tendency was observed in the Mid dose group which was considered to be adverse.
In summary for the reproductive effects, the changes in High dose males were most probably secondary to significant body weight loss during the study. However, in females a significant reduction in implantations was not associated with body weight or food intake effect and as such is probably a reprotoxic effect at the High dose. Death/resorption of embryos/foetuses at the High dose were treatment related, reduced numbers of pups born, relatively high numbers of pups born dead and the death of all High dose pups by Day 3, were considered as treatment related. During gestation, the lower body weight gain at the High dose was probably a reflection of the presence of fewer foetuses. It is not considered likely that the lower body weight of the dams caused to foetal losses. During gestation all High dose pups died, and the dams had a food intake similar to females with no litter; it is not possible to be sure if the low food intake caused low milk production and pup death, or if the lack of pups resulted in less milk production hence a lower food intake, although this was clearly a treatment related effect. In the Mid dose, there were statistically significant differences suggesting a similar trend to the High dose, a dose response could be observed from the data on these parameters.
Test item-related pale discoloration of the liver were observed in 3/11 High dose males and in 7/7 High dose females. The prostate and seminal vesicles were small in 1/11 High dose male, considered as test item-related; seminal vesicles were also small in 1/12 Low dose males, of uncertain relationship with treatment.
Test item-related changes were observed in the parental organ weights of the liver of Mid (~14%) and High dose male animals (~35%). Multifocal/diffuse hepatocellular vacuolation at 90 mg/kg bw/day dose level both in males and females were observed, ascribed to test item, but not considered as adverse. Reduced weights of the prostate and seminal vesicles in High dose males, compared to controls, was probably secondary to the ~8.5% body weight loss in the first week of the study. No adverse test item related changes were observed in the female animal organ weights.
In the parameters measured in adults and pups, there was no evidence for any neurotoxicity and no effects on thyroids or other endocrine systems, and no evidence for immunological effects in immunocompetent organs or tissues.
No test item-related effect, thus no evidence of genotoxic activity was seen in the Mammalian Erythrocyte Micronucleus Test.
The NOAEL for systemic toxicity of the parental generation was considered to be 15 mg/kg bw/day. (based on a strong effect on body weight and food consumption in the High dose animals, and a slight but statistically significant effect in the Mid dose group).
The NOAEL for reproductive effects of the parental generation: 45 mg/kg bw/day (based on marked adverse findings on pregnancy seen only in the High dose group, whereas statistical differences in the Mid dose were consistent with historical control ranges).
The NOAEL for pups’ (F1 generation) development and survival: 15 mg/kg bw/day (based on the death of all High dose pups and based on Mid dose group statistically significant mortality data).
Referenceopen allclose all
Table 1.Parental Body weights
Dose: 0 0.1% 0.5% 1%
Males Generation
Premating (age)
- Initial F0 (7 weeks) 232 238 241 238
F1 (6 weeks) 149 156 144 131*
F2 (7 weeks) 206 217 197 180
- Final F0 506 507 493 479
F1 510 512 492 447*
F2 531 536 492* 467*
Females
Premating
- Initial F0 (7 weeks) 165 163 165 166
F1 (6 weeks) 127 129 121 114*
F2 (7 weeks) 160 162 148 145
- Final F0 206 204 204 206
F1 281 296 271 255*
F2 285 290 271 269*
Gestation
- Initial F0 218 217 216 218
F1 278 294 267 258*
F2 288 291 277 270
- Final F0 361 365 360 350
F1 396 107 379 369*
F2 401 405 392 378*
Lactation
- Initial F0 280 280 275 267
F1 309 319 292 283*
F2 320 317 305 283*
- Final F0 288 294 290 273
F1 304 319 302 293
F2 313 316 314 300
* p < 0.05 (One-way ANOVA variance)
Table 2.Offspring Body weights
Dose: 0 0.1% 0.5% 1%
Males
- Day 0 F1 6.5 6.8 6.4 6.6
F2 6.5 6.7 6.4 6.3
F3 6.7 6.7 6.8 6.1*
- Day 21 F1 15.6 14.7 13.7* 11.5*
F2 17.7 17.8 14.8* 12.4*
F3 19.7 19.9 17.6 13.4*
Females
- Day 0 F1 6.2 6.4 6.1 6.2
F2 6.1 6.4 6.1 6.0
F3 6.4 6.4 6.4 5.8*
- Day 21 F1 15.4 14.74 13.1* 10.8*
F2 16.5 17.1 14.5 11.4*
F3 18.6 19.3 16.0* 12.9*
* p < 0.05 (One-way ANOVA variance)
Extension on the F2 parents to generate an F3 litter generation:
Male fertility indices at the 0.5% and 1.0% dose levels during breeding for F3 litters were significantly higher than that of the control group.
No significant differences from controls were observed for mating, fertility, or gestation indices, days required to mate, or the length of gestation. No parental females aborted or had physical or behavioral abnormalities during F3 gestation and lactation.
Body weights for males were significantly lower than those of controls throughout the treatment period at the 1.0%; dose level, and from Week 3 through the duration of the treatment period for males at t he 0.5%; dose level. Body weights were significantly lower than those of controls for F2 females at the 1.0% dose level throughout the premating phase (except Weeks 0 and 2} , and during Weeks 1, 3 through 6, and 8 for females at the 0.5% dose level.
Gestation body weights were significantly lower than those of controls at the 1.0% dose level throughout gestation. Body weights during lactation were significantly lower on Days 0, 7, and 14 at the 1.0% dose level; mean body weight on lactation Day 21 at the 1.0% dose level also was lower than that of the control, although this difference was not statistically significant.
Body weight gains were significantly lower than these of controls during weeks 4, 5 through 8, 13, 15, 17 , 20, 23, and 24 formales at the 1.0% dose level , during Weeks 4, 6, 7, 13, 16, 23, and 24 for males at the 0.5% dose level, and during Week 3 for males at the 0.1% dose level. There were no consistent significant differences in weight gains for females during the treatment period.
Body weight gains during lactation were significantly higher than these of the control between Days 14 and 21 at the 1.0% dose level, and between Days 0 and 21 for the 0.5% and 1.0% dose levels.
Food consumptions were significantly lower than those of controls during Weeks 2, 5 through 7, and 10 for males at the 1.0% dose level, and during Weeks 5 through 7 for males at the 0.5% dose level, Female food consumptions were significantly lower than those of controls during Week 2 at both the 0.5% and 1.0% dose levels, and were significantly higher during Week 7 at the 0.1% dose level.
F3 Litter data:
No significant differences from controls were observed for the mean number of pups born alive, litter size, survivability, or sex ratio. The proportions of the total number of pups born alive and found dead at the 1.0% dose level were significantly lower and higher, respectively, than those of the controls. These differences may merely reflect the effect of a slight reduction in the number of litters in the control group, and a disproportionate influence of very few litters in the high-dose group; i.e. , two litters accounted for 13 (six plus seven) of the 17 pups found dead; there was one dead pup each in four other litters. Male and female pup weights and pup weight gains at the 1.0% dose level were significantly lower than these of controls throughout lactation. At the 0.5% dose level, pup weight gains for males and females from Days 4 through 7, for males from Days 7 through 14, and tor fema1es from Days 14 through 21 were significantly lower than these of the control, as were male pup weights on Day 14 and male and female pup weights on Day 21. One pup in the0.5% dose group and 17 pups from seven litters In the 1.0% dose group had no milk in the abdomen on Day 4 of lactation. One female pup in the control group was icteric on Day 4. Another control pup from a different litter was missing the left eye. Five males and five females from one litter at the 1.0% dose level had urine stains on Day 21.
No test item related mortality was observed in the study.
One Control male (#1009) was found dead on Day 28. No clinical sings were recorded prior to death. The cause of death could not be determined.
One High dose male (#4004) was preterminally euthanised on Day 14. Hunched back, piloerection, and wasted were observed prior to euthanasia. The cause of the death was the poor clinical condition, which was probably treatment related, but no cause of the clinical effects was evident from the macroscopic or histological evaluation. One Low dose female (#2510) was preterminally euthanised on PND0 because of prolapsed vagina, considered to be unrelated to treatment. Moderate paleness (whole body) and piloerection were recorded prior to death.
-clinical observations:
No test item related clinical signs were observed in the study.
Liquid faeces were recorded during the lactation period (Day 52) for one Low dose female (#2503) and on Day 51 for a Mid dose female (#3505).
Red liquid in the vulva was observed for one High dose female (#4509) on Day 33 and 34. Piloerection was observed for this animal on Day 16.
Noisy respiration was observed for one Control female (#1503) on Day 6 and 7.
Hunched back and piloerection were observed for one Control female (#1510) form Day 17 to Day 20.
These findings were all considered as being incidental, not related to the test item administration.
-body weight and body weight gain:
Test item related adverse effect was observed on body weight gain parameters in High dose (90 mg/kg bw/day) males and females and Mid dose females (45 mg/kg bw/day). No test item related adverse effect on body weight or body weight gain was detected in Mid dose (45 mg/kg bw/day) male or Low dose (15 mg/kg bw/day) animals (males or females).
In the first week, High dose males lost 8.5% of their bodyweight, with weight gain thereafter. This High dose body weight loss, in the first week, was considered as a test item related effect.
No effect on body weight parameters was seen in Mid and Low dose males.
In case of females, the observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period in all dose groups . The Mid and High dose females showed reduced body weight / body weight gain at the end of the gestation period (body weight was lower by 7.8% (p<0.05) and 16.7% (p<0.01), body weight gain by 21.8% (p<0.01) and 42.2% (p<0.01)).
In case of High dose females, statistically significantly lower body weight parameters were recorded by the end of the lactation period (by 19.5% (p<0.01) lower body weight and by 87.4% (p<0.01) lower body weight gain), with virtually no weight gain at all during the lactation period. The body weight gain from Day 0 to PPD 13 was statistically significantly lower for Mid dose and High dose females than Control (by 14.8% (p<0.05) and 70.6% (p<0.01)). Body weight data from PND 7 in the High dose were outside of the historical control range. These body weight effects were considered as being a test item related adverse effect.
No effect on body weight parameters was seen in Low dose females.
-food consumption:
Test item related adverse effects were observed on food consumption in High dose males and females (90 mg/kg bw/day) and Mid dose females (45 mg/kg bw/day) during the treatment period; Mid and Low dose males and Low dose females were not affected.
Statistically significantly decreased (p<0.01) food consumption was observed in the High dose male group (90 mg/kg bw/day) at an average of ~30% below control for the 4-week period. The other groups (Mid and Low) were unaffected.
Statistically significantly decreased food consumption was observed in the Mid (p<0.05) and High dose (p<0.05 and p<0.01) female groups during gestation and lactation periods. Premating the female food intakes were not significantly different to control (all ~11-15g/day); during gestation although there were statistical differences, all groups were within about 15% of the controls; during lactation normal control food intake goes up dramatically (from about 35g/day initially, up to ~70g/day by Days 10-13) but the High dose group had no normal increased food intake and remained at the pre-lactation range of generally <20g/day, the Mid dose group were generally within 15-20% of the control values, which is a relatively slight effect. Together with the body weight data, the observed values were considered as a test item related adverse effect, but at the High dose the very low food intake could not support a normal high-energy-requirement for the level of lactation needed for pup growth.
-neurological assessment:
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength. There was statistically significantly increased (p<0.05) landing splay (Hind paws) observed in the High dose male group, but the data were within the historical control range therefore these changes did not consider as test item related effects. There was statistically significantly increased (p<0.05) landing splay (Fore paws) observed in the Low and Mid dose male groups.
All dose groups of males and females had comparable locomotor activity to the Control. There was statistically significantly increased LMA distance (10-15 min) was observed in the Low dose male group without dose response. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Haematology
There were statistically significantly changes in the male dosed groups sporadically, but all the data were within the historical control range therefore they were not considered as test item related effects. There was statistically significantly decreased reticulocyte relative (%) and mean cell volume observed in the High dose female group and the values were outside of the historical control range, hence it was considered as test item related effect.
-Clinical chemistry
There were test item related effects observed in the High dose male and female groups on clinical chemistry parameters. There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration in the Low dose group for both sexes.
Statistically significantly decreased cholesterol concentration was observed in Mid dose male and High dose males and females (p<0.01), the data were outside of the historical control range. But this fact was most probably related to the significantly reduced food consumption of animals during the gestation and lactation periods, not considered as being a direct adverse effect.
Statistically significantly decreased total protein (p<0.01) was measured in the High dose male group (possibly related to food intake) but the data were within the historical control range.
Statistically significantly increased A/G ratio (p<0.05) was measured in the High dose male group, the data were outside of the historical control range, hence it was considered as test item related effect but probably related to food intake. There was statistically significantly increased (p<0.01) bile acid concentration observed in the High dose male group, the data were outside of the historical control range, therefore it was considered as a possible test item related effect. Statistically significantly increased chloride concentration (p<0.01) and decreased phosphorus concentration (p<0.05) was observed in the High dose female group, but the data were within the historical control range.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the Mid dose males (p<0.01) and High dose males (p<0.01) and females (p<0.01) when compared to control animals. In case of High dose females, the observed values were outside of the historical control range. Furthermore, increased Alkaline phosphate (ALKP) activity was recorded in the Mid dose males and females (p<0.01) and High dose males and females (p<0.01), the observed female mean values were within of the historical control range, the High dose male value was outside of the historical control range.
Statistically significantly increased urea concentration (p<0.01) was observed in the Mid dose female groups, which was outside of the historical control range but without dose response it was not considered as test item related effect. Statistically significantly increased potassium and calcium concentration (p<0.05) and was observed in the Mid dose female groups, which were within the historical control range.
Observed values of alanine aminotransferase (ALT/GPT), A/G (albumin/globulin) ratio and bile acid concentration in case of the High dose males were considered as test item related but none were of a magnitude to be considered clearly adverse. These changes were probably related to the low food intakes and/or the histopathology findings in the liver.
-Urinalysis
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urinary blood (p<0.01) was observed in the High dose male group but in the absence of any histopathological kidney changes are not considered as a clear adverse effect of the test item. Statistically significantly decreased specific gravity (p<0.01) and protein level (p<0.05) was observed in the High dose female group. The specific gravity data were outside of the historical control range. Statistically significantly increased urine crystals (p<0.01) were observed in the High dose female group.
-oestrus cycle evaluation in females
Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00-4.04 days was observed in the different groups) before starting the treatment period.
Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.08, 4.00, 4.02 and 4.11 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in 1 Mid dose female and 2 High dose females were affected (#3510, #4506 and #4509), but as it did not affect mating or pregnancy, this fact was considered as being an occasional finding, not being a test item related effect.
Prolonged dioestrus was not noted.
Pseudo-pregnancy was noted for one Mid (#3509) dose female, but this frequency was in line with the normal, expected range.
-reproductive ability assessment and indices
There were test item related adverse effects for the High dose group with regard to reproductive ability or gestation indices. The mating index was 100% in all groups (males and females). The fertility index was 100%, 92%, 100% and 91% in the Control, Low, Mid and High dose groups for males and 100%, 92%, 100% and 92% in the Control, Low, Mid and High dose females. The gestation index was 100%, 100%, 100% and 55% in the Control, Low, Mid and High dose females.
Eleven females in the High dose group were pregnant. However, one female had no live born pups (#4502) and four pregnant females (#4507, #4509, #4510 and #4512) had no delivery. Corpora lutea and implantations were observed for these 4 females but 100% of the embryos were resorbed. It was considered as an adverse test item related effect, the number of females with complete resorption of embryos was well outside the historical control range.
There were statistically significantly decreased (p<0.05 and p<0.01) number of corpora lutea and pre-implantation loss in the Mid and High dose groups which were considered as test item related adverse effect.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation). The mean duration of mating was 2.58, 2.33, 2.33 and 2.25 days in the Control, Low, Mid and High dose groups, respectively.
-evaluation of the gestation, parturition and post-partum period:
There was effect of treatment noted during the gestation period, parturition and post-partum period in the Mid and High dose groups.
The mean duration of pregnancy was statistically significantly increased in the High dose group, the data were outside of the historical control range.
As far as it could be observed during the study, the parturition was normal for all animals, except one Low dose female (#2510), one Mid dose female (#3511) and two High dose females (#4502 and #4504).
The number of implantation sites was statistically significantly lower (p<0.01) in the High dose group compared to the Control, although the food intake was normal up to the time of implantation (~ GD7).
There were statistically significant differences that could be ascribed as treatment related on pre-natal, post-natal or total mortality values (litter mean and %) in the Mid and High dose groups compared to the Control. For High dose group the post-natal mortality and total mortality data were 100% which is far higher than historical control groups. The total intrauterine mortality value in the Mid dose group was outside of the historical control group range. For the High dose, the very low food intake by the dams would have precluded an adequate milk production, low growth and death of pups would be expected as secondary to the food intake effect. In the Mid dose a statistically lower food intake by the dams may have influenced the pup growth and mortality data.
-thyroid hormone analysis
There was no test item related effect on the thyroid hormone concentration levels recorded in the PND13 pups and for the males.
The terminal body weight of the pups was lower compared to the Control therefore the thyroid gland weights of the PND13 pups were lower as well, but the thyroid weight adjusted for body weight was unaffected. There was statistically significantly decreased only for the absolute pup thyroid gland weight compared to the Control. In summary, there were considered to be no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
Statistically significantly decreased T4 concentration was measured in the Low dose group but the data were within the historical control range.
-organ weights:
Parental Males
Test item-related changes were observed in the organ weights of the liver of Mid and High dose male animals and in the prostate and seminal vesicles in High dose males compared to controls.
Statistically significantly decreased kidney (absolute (p<0.05) and brain related (p<0.01)) weight was observed in the High dose male group, but not when adjusted for body weight hence it was considered as not a test item related effect.
Statistically significantly increased liver (absolute, body and brain related (p<0.01)) weight was observed in the High dose male group and body related liver weight in the Mid dose group. The absolute and relative to body weight of the liver in the High dose group were outside of the historical control range and these statistically significant weight changes were correlated with macroscopic and microscopic findings therefore they were considered as test item related effect.
Statistically significantly decreased prostate gland (absolute, body and brain related (p<0.05)) weight was observed in the High dose male group, the data were within the historical control range, but based on the histopathological changes, the weight difference is considered to be adverse but probably a result of low food intake and ~10% body weight loss in the first 7 days of the study.
The relative to brain weight of the seminal vesicle decreased by -19.8% in High dose males compared to controls. The data were within the historical control range, but based on the histopathological changes, the weight difference is considered to be adverse but probably a result of low food intake and ~10% body weight loss in the first 7 days of the study.
The other statistically significant organ weight changes were within the historical control range and without histological relevance and were considered as incidental.
Parental Females
Statistically significant organ weight changes were seen in the brain, heart, kidney, spleen, uterus and ovary. The data did not show dose-relation and/or were without histological relevance therefore they were considered as incidental.
Statistically significantly decreased body weight of the High dose females was observed, the data were outside of the historical control range.
Statistically significantly increased brain weight related to body was observed in the High dose female group. The data were considered to be due to lower body weight and were without histopathology findings, it was not considered as test item related adverse effect.
Statistically significantly decreased absolute, body and brain related (p<0.01) weight of the heart was measured in the High dose female group and statistically significantly decreased relative to brain and absolute weight was measured in the Mid dose group. The absolute and brain related values were outside of the historical control range. However, in the absence of any histopathological cardiac changes, the weight difference is not considered to be adverse.
Statistically significantly decreased kidney (absolute (p<0.01) and brain related (p<0.01)) weight was observed in the High dose female group, but not for the body weight relative mean. Since these rats were smaller, lower kidney weights are expected, also there were no histopathological changes, hence this kidney weight difference is not considered to be adverse.
Other statistical differences did not show dose-relation, were a reflection of lower body weight at the High dose and/or were without histological relevance and were considered as incidental.
-pathology evaluation:
NON-PREGNANT FEMALES / Parental Generation
Two females (Low dose #2512 and High dose #4505) were non pregnant during the study.
At necropsy no test item-related changes were seen in these females or their male pair, in the female #4505 the ovaries were small, the uterine horns were dilated and were considered as incidental, in female #2512 no macroscopic findings were noted. In both males (#2012 and #4005) the mandibular lymph nodes were dark red and were considered as procedure related (due to sublingual blood sampling).
Microscopically in the #4505 female ovarian atrophy and dilatation of uterine horns were seen and considered to be incidental, in the male pair (#4005) of this female, minimal multifocal hepatocellular vacuolation, and minimal decreased secretion in the prostate and seminal vesicle were present (similarly to the terminal animals) which were considered as test item-related. Other findings in these two animals were considered procedure related, incidental or a common background.
In the #2512 female no microscopic findings were noted, in the male pair (#2012) of this female sinusoidal erythrocytosis was present in the mandibular lymph nodes, as procedure related (due to sublingual blood sampling).
FOUND DEAD ANIMAL / Parental Generation
One Control male (#1009) died on Day 28 of the study. The cause of death could not be determined.
Macroscopic Findings
The lungs, the thymus and the liver were dark red, focal red discoloration was seen in the glandular mucosa of the stomach.
Microscopic Findings
Microscopically congestion/haemorrhage in the lungs and thymus; these were considered as agonal, or incidental.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Low Dose female (#2510) was preterminally euthanised on Day 37 (after giving birth) due to prolapsed vagina. One High Dose Male (#4004) was preterminally euthanised on Day 14 of the study due to weak general condition.
Macroscopic Findings
Prolapsed vagina and invagination of the uterine horn was seen at necropsy for the Low dose female; there were no significant findings in the male.
Microscopic Findings
No test item-related changes were observed microscopically for the Low dose female. In case of the High dose male the mandibular lymph nodes were dark red, the thymus was small, on the glandular gastric mucosa white and red foci were present at necropsy, microscopically and were considered as not test item related. The cause of termination of the male was considered to be treatment related clinical signs, but there was no evidence of pathological changes that could indicate the aetiology of the adverse clinical signs.
TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Test item-related pale discoloration of the liver were observed in 3/11 High dose males and in 7/7 High dose females. The prostate and seminal vesicles were small in 1/11 High dose male, considered as test item-related; seminal vesicles were also small in 1/12 Low dose males, of uncertain relationship with treatment.
All other changes were considered procedure related, incidental or a common background.
Microscopic Findings
Test item-related multifocal/diffuse (mainly multifocal) hepatocellular vacuolation was detected in 10/11 High dose, in 4/12 Mid dose males and in 7/7 High dose and in 7/12 Mid dose females with minimal to marked severity (typically minimal/mild severity in males and moderate in the females), correlated with organ weight changes and necropsy findings.
In High dose males, in the prostate decreased secretion was seen in 7/11 cases, in the Mid dose in 2/12 cases with minimal/mild severity. Decreased secretion was detected in the seminal vesicles in 4/11 High dose males (correlated with organ weight changes) and were considered as test item related. Decreased secretion was detected in the seminal vesicles in 1/9 Low dose male as well and was considered as incidental. Similar findings in secondary sex organs are common when animals have a significant body weight loss in the first week of treatment.
In the kidney of all examined High dose males, minimal multifocal eosinophilic droplets/globules were seen in the renal tubules and were considered as test item related. These hyaline droplets are most likely to be Alpha 2u globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only; in the context of human safety evaluation, this rat-specific change is considered to be non-adverse.
All other findings were considered as incidental or background and not to represent adverse effects of the test item.
There were test item effects on mortality or survival of the pups (F1 generation) in the Mid and High dose groups, associated with low food intake by the dams during lactation.
The mean number of viable pups born were statistically significantly decreased in the Low (p<0.05) and High dose groups (p<0.01). Pre-natal mortality for the Mid (p<0.05) and High (p<0.01) dose groups was statistically significantly increased compared to the Control. The Mid and High dose groups values were outside of the historical control range.
Total mortality in the High dose groups was outside of the historical control data and clearly an adverse effect (probably related to the very low food intake that was insufficient for adequate lactation). A similar trend in the Mid dose was statistically significantly increased compared to the control but was generally similar to the Low dose group where there were no statistical differences to control.
Evidence of suckling was recorded for all live born pups in the study except in case of two High dose dams (#4503 and #4506), where the pups were born alive but shortly after the end of the delivery were seen to have died (which was clearly not related to low milk production subsequent to low food intake but was considered to be an adverse effect of treatment).
Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups.
-Body weight and body weight gain:
There were test item related statistical differences in the offspring body weights or weight gains in the Mid dose group when compared to the controls, in the High dose group, the limited available data also indicates a weight growth effect. The measured values were within the historical control range for the Mid dose throughout the development phase.
Because of the lower maternal food consumption by 15-20% in the Mid dose, less milk and lower growth of the pups was observed, therefore this effect is considered as secondary to maternal effect. In the High dose, the 100% mortality prevents a proper assessment of pup growth.
-Anogenital distance, nipple retention:
No test item effect was observed on anogenital distance or nipple retention during the study.
There were statistically significantly decreased anogenital distance values observed in the Mid and High dose male groups when compared to the Control, but all the data were within the historical control range and when adjusted for the cube root of body weight, therefore these effects were not considered as test item related.
There was no nipples/areolae presence seen in any of the male pups on PND13.
-Thyroid hormone analysis:
There was no test item related effect on the thyroid hormone concentration levels recorded in the PND13 pups.
The terminal body weight of the pups was lower compared to the Control therefore the thyroid gland weights of the PND13 pups were lower as well, but the thyroid weight adjusted for body weight was unaffected. There was statistically significantly decreased only for the absolute pup thyroid gland weight compared to the Control. In summary, there were considered to be no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
-Pathology:
TERMINAL / F1 Generation (PND13)
Macroscopic Findings
No test item-related macroscopic findings were observed up to the dose level of 45 mg/kg bw/day. The High dose pups all died, most of them were autolysed or cannibalised so examinations, but of the observable tissues and organs there were no trends suggesting the presence of a treatment related effect visible at necropsy.
Microscopic Findings
No histopathological examination was performed on pups (F1 generation).
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 45 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Klimisch 1
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Reproductive screening
A key subacute OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (propylene glycol), 15, 45 and 90 mg/kg bw/day (Szalóki, 2022). Based on the results of the DRF study, 90 mg/kg bw/day was selected as the High dose for this study. Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT). Results of this MNT are summarized in Section 7.6.
The systemic toxicity parameters are reported under the Section 7.5. The reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND (Post-natal Day) 13 pups and parental males were also determined. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, the retained reproductive organs of all Control and High dose animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved.
Eleven females in the High dose group were pregnant. However, one female had no live born pups and four pregnant females had no delivery (100% of the embryos was resorbed). High dose pups who were live born were dead in three days after birth the latest. It was considered as test item related adverse effect. There were severe test item related effects on pup mortality or survival of the pups (F1 generation) in the High dose groups; slight but similar tendency was observed in the Mid dose group which was considered to be adverse. In summary for the reproductive effects, the changes in High dose males were most probably secondary to significant body weight loss during the study. However, in females a significant reduction in implantations was not associated with body weight or food intake effect and as such is probably a reprotoxic effect at the High dose. Death/resorption of embryos/foetuses at the High dose were treatment related, reduced numbers of pups born, relatively high numbers of pups born dead and the death of all High dose pups by Day 3, were considered as treatment related. During gestation, the lower body weight gain at the High dose was probably a reflection of the presence of fewer foetuses. It is not considered likely that the lower body weight of the dams caused to foetal losses. During gestation all High dose pups died, and the dams had a food intake similar to females with no litter; it is not possible to be sure if the low food intake caused low milk production and pup death, or if the lack of pups resulted in less milk production hence a lower food intake, although this was clearly a treatment related effect. In the Mid dose, there were statistically significant differences suggesting a similar trend to the High dose, a dose response could be observed from the data on these parameters. In the parameters measured in adults and pups, there was no evidence for any neurotoxicity and no effects on thyroids or other endocrine systems, and no evidence for immunological effects in immunocompetent organs or tissues. The NOAEL for reproductive effects of the parental generation: 45 mg/kg bw/day (based on marked adverse findings on pregnancy seen only in the High dose group, whereas statistical differences in the Mid dose were consistent with historical control ranges). The NOAEL for pups’ (F1 generation) development and survival: 15 mg/kg bw/day (based on the death of all High dose pups and based on Mid dose group statistically significant mortality data).
Mulitigeneration testing
Further data on reproductive toxicity were available from read across substance Docusate sodium (CAS No. 577-11-7).
Justification for read across with the category of di-ester sulfosuccinates is documented in a separate document attached in Section 13.
- A key 3-generation toxicity study at dietary dose levels of 0.1, 0.5 and 1.0% in the diet (MacKenzie, 1986) conducted according to OECD caused a reduction in body weights at the dose levels of 0.5 and 1% in the diet for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations, however this did not interfere with growth and development or reproductive performance, and had no adverse effects at levels on the reproductive function of either sex in any generation up to 1%. There were no other effects on parental or reproductive parameters. The NOEL for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0%, which was considered to correspond with approximately 750 mg/kg bw/day.
- In a supporting 2-generation toxicity study in rats, 0.5 and 1% were given in the diet (Levinskas & Shaffer, 1970). In the first mating of the F0 generation and the second mating of the F2 generation, pups were weaned directly onto the diets which were being fed to their parents. In the other 3 matings of this study, dams were given a control diet on the day before delivery to avoid a bitter taste of the milk. Pups of all litters were examined for gross defects. Autopsies were performed, however, only on pups from the first mating of the F2 animals. Portions of all major organs were taken for histopathology processing and examination from one male and female from each litter. The other male and female were skinned and eviscerated, and the carcasses cleared, and the skeletons stained and examined for defects. In both the first mating of the F0 generation and the second mating of the F2 generation, the fertility and gestation indices were high and comparable. The viability index was good, albeit slightly down for the F3b pups, while the lactation index was depressed for both of these matings. In addition, the mean weight of the pups at weaning decreased with increasing concentrations of test material in the diet of the dams. In the second mating of the F0 animals, the viability and lactation indices and the mean weight of the test pups at weaning still showed decreases relative to the control values. However, in the 2 subsequent matings, all indices for the dosed animals were numerically high and compared favorably with the corresponding control values. Also, the mean weight of the pups at weaning was essentially similar for all groups. Consequently, it is concluded that diets containing 1% or less had no adverse effect on the reproduction and lactation performance of rats. The lowering of the survival rate and the mean body weight of the F1a and F3b pups is attributed to an impairment of nutrition as a result of the taste which is believed to have been secreted into the milk of the dams. Microscopic study of tissues showed findings which were similar in all groups. In processing the skeletons, the presence of an extra sternebrae in the sternum between the 5th and 6th sternebrae was not considered to parental exposure of test material. It is concluded that feeding of test material to rats from weaning through reproductive age for successive generations at levels of 1%, or less, did not produce lesions or anomalies in the offspring which could be attributed to the compound.
In conclusion, 2 multigeneration studies with read-across substance CAS no. 577 -11 -7 (Docusate sodium) showed that there were no reproductive findings.
Conclusion
A combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD No. 422) in rats with the registered substance showed a NOAEL for reproductive effects of the parental generation of 45 mg/kg bw/day (based on marked adverse findings on pregnancy seen only in the High dose group, whereas statistical differences in the Mid dose were consistent with historical control ranges). In addition, the NOAEL for pups’ (F1 generation) development and survival was 15 mg/kg bw/day (based on the death of all High dose pups and based on Mid dose group statistically significant mortality data).
Multigeneration studies with read across substance Docusate sodium (CAS 577 -11 -7) showed slight maternal/paternal toxicity at 0.5 and 1% in the diet, however this was not confirmed in the second study. From both studies, it can be concluded that the substance up to 1% in the diet did not lead to effects on fertility or postnatal development; this concentration corresponds with 750 mg/kg bw/day, which is higher than the NOAEL for paternal/maternal toxicity. Based on the secondary findings in the screening study, and absence of findings in the repeated dose studies and the multigeneration studies with structurally related similar subatnces no further testing is needed.
Effects on developmental toxicity
Description of key information
A new Prenatal developmental toxicity study with the registered substance is planned and will be updated later when results are available (currently waived in the dossier).
A combined reproduction-teratogenicity study with read across substance CAS No. 37294-49-8 (Disodium C-isodecyl sulphonatosuccinate) in 2 generations resulted in a NOAEL for embryotoxicity of 750 mg/kg (1% in the diet) and a NOEL for teratogenicity of 3000 mg/kg (4% in the diet).
Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw, whereas at 2% in the diet visceral and skeletal anomalies were observed, which was secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed.
Based on the absence of developmental findings in the screening study and teratogenicity study, no further testing is needed.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1976
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- only 2 doses tested. Allthough not all details on the study design were provided, the study was performed to the highest standards at the time of conduct. In the current study, a concurrent test article, dioctyl calcium sulfosuccinate (DCS) was also tested at 0.5, 1, 1.5 and 2% in the diet.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: About 2 months
- Weight at study initiation: Not provided
- Fasting period before study: Not provided
- Housing: Individually in hanging wire mesh cages
- Diet (e.g. ad libitum): Wayne Lab Meal ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Not provided
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2°C
- Humidity (%): 50 ± 5%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: feed
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: 40% solution in corn oil
DIET PREPARATION
- Rate of preparation of diet (frequency): Not provided
- Mixing appropriate amounts with (Type of food): 1.0% and 2.0% admixed in Wayne Lab Meal
- Storage temperature of food: Not provided
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 40% solution in corn oil
- Amount of vehicle (if gavage):/
- Lot/batch no. (if required): Not provided
- Purity: Not provided - Details on mating procedure:
- Not provided
- Duration of treatment / exposure:
- gestational days 6 through 15: dosing
- Duration of test:
- gestational days 6 through 15: dosing
gestational day 21: killing of the mothers and removing fetuses by cesarean section - No. of animals per sex per dose:
- 22 female rats in dose 1.0% DSS
20 female rats in dose 2.0% DSS - Control animals:
- yes, concurrent vehicle
- Details on study design:
- Not provided
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes (clinical condition and signs of illness)
- Time schedule: each day
- Cage side observations were not included.
BODY WEIGHT: Yes
- Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: Ovaries and uterine content - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes - Fetal examinations:
- - External examinations: Yes (3336 of conceptuses)
- Soft tissue examinations: No
- Skeletal examinations: Yes (one half of the total number of fetuses)
- Head examinations: No
-Visceral examinations: Yes (one-half of the total number of fetuses were fixed in Bouin’s fluid for a detailed examination of visceral anomalies, using the slicing method of Wilson) - Statistics:
- Maternal body-weight gains, maternal food consumptions and fetal weights were analyzed by Dunnett’s two-sided, multiple comparison test. Frequencies of resorptions and fetal abnormalities among litters were analyzed by the Mann-Whitney U test or the Chi-square test (with Yate’s correction), as appropriate.
- Dose descriptor:
- NOAEC
- Effect level:
- 1 other: %
- Based on:
- act. ingr.
- Remarks:
- in the diet
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEC
- Effect level:
- 1 other: %
- Based on:
- act. ingr.
- Remarks:
- in the diet
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 074 mg/kg bw/day (nominal)
- Based on:
- act. ingr.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 074 mg/kg bw/day (nominal)
- Based on:
- act. ingr.
- Basis for effect level:
- other: developmental toxicity
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 074 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: no remarks
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 074 mg/kg bw/day
- Based on:
- act. ingr.
- Sex:
- female
- Basis for effect level:
- other:
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Developmental effects observed:
- not specified
- Conclusions:
- Subtoxic dietary levels of 1.0% docusate sodium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. Interpretation of the results of the present experiments, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.
- Executive summary:
Prenatal developmental toxicity was studied in rats dosed from day 6 to day 15 of gestation by dietary administration of docusate sodium at dose levels of 1.0 and 2.0 % in the diet.Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared the controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophtalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. There were significant depressions in maternal weight gains in the 2.0% DSS-group . Interpretation of the results of the present experiment, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.
The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with 1074 mg/kg body weight, as calculated in the study.
Reference
TABLE 1. Maternal and fetal results of pregnant rats given various amounts if DSS in their diets during
gestational days 6 through 15.
Parameter |
Dose: Control |
1.0% DSS |
2.0% DSS |
Maternal |
Groups: (I-A) |
(II-A) |
(II-B) |
No. of pregnant rats |
43 |
22 |
20 |
No. of pregnancies with total resorptions |
0 |
0 |
1 |
No. of pregnancies with viable fetuses |
43 |
22 |
19 |
Average weight gain of dams with viable fetuses(g): |
|
|
|
Days 6 to 15 |
78 |
86 |
52* |
Days 15 to 21 |
66 |
67 |
77 |
Avarage, apparent food intake of dams with viable fetuses (g/rat/day): |
|
|
|
Days 6 to 15 |
22.5 |
24.8 |
21.4 |
Days 15 to 21 |
28.6 |
32.1 |
33.4 |
Calculated compound consumed (mg/kg/day) |
-- |
1074 |
1988 |
Litters |
|
|
|
Total number of: implantations |
411 |
203 |
219 |
Resorptions (% occurence) |
23 (5.6) |
8 (3.9) |
30*a (13.7) |
Dead fetuses (% occurrence) |
3 (0.7) |
0 |
1 (0.5) |
Viable fetuses (% occurrence) |
385 (93.7) |
195 (96.1) |
188 (85.5) |
Fetal weight (g) |
4.6 |
5.2 |
4.7 |
Litters size (viable fetuses) |
8.9 |
8.9 |
9.9 |
External major malformations1: No. of litters affected (% occurrence) |
0 |
0 |
5* (25.0) |
No. of fetuses affected (% occurrence) |
0 |
0 |
36*a (20.2) |
* Significantly different from control (p< 0.05)
a Significance by Chi-square, but not Mann-Whitney U test
1 Primarily, exencephaly varying degrees and associated anomalies (See TABLE 2)
TABLE 2. Morphological observations of fetuses delivered from rats given DSS in their diets on
gestational days 6 through 15.
Morphology |
Dose: Control |
1.0% DSS |
2.0% DSS |
External observations1: |
Groups: (I-A) |
(II-A) |
(II-B) |
Total number examined |
388a |
195 |
189 |
Major anomalies: Adactyly |
0 |
0 |
0 |
Hemimelia |
0 |
0 |
0 |
Schistocelia |
0 |
0 |
2 |
Dome shaped head |
0 |
0 |
0 |
Cranial bubble (1-2mm) |
0 |
0 |
9* |
Exencephaly |
0 |
0 |
18* |
Exencephaly (cleft condition) |
0 |
0 |
7* |
Anencephaly |
0 |
0 |
0 |
Spina bifida |
0 |
0 |
6 |
Macroglossia |
0 |
0 |
0 |
Micro- or anophtalmia |
0 |
0 |
3 |
Defects: Hematoma (subcutaneous) |
2 |
0 |
0 |
Edamatous abdomen |
0 |
0 |
0 |
Tail short & curled |
0 |
0 |
0 |
Abducted fifth digit, left Rear foot |
0 |
0 |
1 |
1 Fetuses may have more than one defect
a Fifty-four fetuses examined grossly only. (Shipment c valid as controls only)
*Significantly different from control (p< 0.05) by Chi-square only
TABLE 3. Visceral observations of fetuses delivered from rats given DSS in their diets on gestation days
6 through 15.
Visceral observations |
Dose: Control |
1.0 % DSS |
2.0% DSS |
Groups: (I-A) |
(II-A) |
(II-B) |
|
Total number of fetuses examined |
165a |
98 |
91 |
Defects1: Exencephalous characteristics |
0 |
0 |
11* |
Dilated lateral ventricles |
1 |
3 |
5 |
Microphtalmia |
0 |
1 |
0 |
Anolphtalmia |
0 |
0 |
23* |
Retinal foldings |
0 |
0 |
0 |
Anotia or microtia |
0 |
0 |
0 |
Cleft palate |
0 |
0 |
1 |
Situs transversus – aorta, esophagus & stomach |
1 |
0 |
0 |
Intestinal agenesis |
0 |
0 |
0 |
Arch of aorta absent or right sided |
0 |
0 |
0 |
Diaphragmic hernia |
0 |
0 |
1 |
Dilated renal pelves |
2 |
0 |
3 |
Ectopic kidneys(s) &/or variation in size |
1 |
0 |
0 |
Renal agenesis |
0 |
0 |
2 |
Dilated ureters |
6 |
0 |
3 |
Adrenal agenesis |
0 |
0 |
1 |
Testes – ectopic or enlarged |
1 |
0 |
1 |
Hermaphroditism |
0 |
0 |
3 |
1Fetuses may have more than one defect
aExcludes 1 fetus lost
*Significantly different from control (p<0.05) by Chi-square only
TABLE 4. Skeletal observations of fetuses delivered from rats given DSS in their diets on gestation days
6 through 15.
Skeletal observations |
Dose: Control |
1.0 % DSS |
2.0% DSS |
Groups: (I-A) |
(II-A) |
(II-B) |
|
Total number of fetuses examined |
167a |
97 |
98 |
Defects1: Cranial bones, incomplete to lack of ossification : Nasal |
0 |
0 |
4 |
Frontal |
1 |
0 |
20* |
Parietal |
1 |
1 |
19* |
Interparietal |
1 |
2 |
18* |
Supraoccipital |
0 |
0 |
15* |
Exoccipital |
0 |
0 |
2 |
Atlas |
0 |
0 |
1 |
Zygomatic |
0 |
0 |
1 |
Premaxilla |
0 |
0 |
1 |
Tympanic bullae |
0 |
0 |
5 |
Mandibles |
0 |
0 |
1 |
Hyoid |
0 |
0 |
3 |
Eye orbit, reduction |
0 |
0 |
0 |
Exoccipital, fused to atlas |
0 |
0 |
0 |
Vertebrla column, curved &/or open |
0 |
0 |
5 |
Vertebrae: |
|
|
|
misshapened &/or retarded development |
0 |
0 |
5 |
thoracic, bipartite centra |
2 |
1 |
5 |
lumbar, bipartite centra |
0 |
0 |
2 |
Sternebrae: |
|
|
|
fused |
0 |
0 |
0 |
hypoplastic to absent |
0 |
0 |
1 |
one or two absent |
1 |
0 |
0 |
staircase |
0 |
0 |
3 |
bipartite |
0 |
0 |
2 |
Rib(s): |
|
|
|
accesory |
6 |
5 |
5 |
Absent or less developed |
0 |
0 |
7* |
wavy |
2 |
2 |
0 |
fused |
0 |
0 |
2 |
Pelvic, hypoplastic to absent |
0 |
0 |
0 |
Brachydactyly |
0 |
0 |
0 |
Syndactyly |
0 |
0 |
0 |
Adactyly |
0 |
0 |
0 |
Hemimelia & small scapula |
0 |
0 |
0 |
1Fetuses may have more than one defect
aExcludes 1 fetus destroyed during cleaning process
*Significantly different from control (p<0.05) by Chi-square only
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 074 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Klimish 2
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Supporting data for absence of teratogenicity and developmental toxicity was available from following studies
- Combined reproduction-teratogenicity study with read across test item CAS No. 37294-49-8 ( Disodium C-isodecyl sulphonatosuccinate) in 2 generations (Tegeris and Underwood, 1975c). The test item containing +-50% active ingredient was fed to Charles River CD Sprague-Dawley rats at 1% and 4% in the diet, while the control group received normal diet. The original females were allowed to deliver their first 2 litters (F1a & F1b), while the third litters were used for teratological evaluation and partial histology on 5 pairs per group (F1c). Females were allowed to rest 20 days between weaning and their next breeding. All litters were standardized to 8 newborn to equalize the maternal stress.
-F1a pups were examined to calculate Fertility Index (FI), Viability Index (VI) and Lactation Index (LI); they were discarded at weaning.
- F1b pups were examined to calculate Fertility Index (FI), Viability Index (VI) and Lactation Index (LI), with part of them that were studied postmortem (autopsy and 5 pairs per group for histology) and the other part were selected for second generation breeding, leading to F2a (discarded at weaning) and F2b (teratological examination and partial histology on 5 pairs per group).
-F1c were used for teratological evaluation an d partial histology on 5 pairs per group.
Although data suggest that the test material under the conditions of this experiment is not teratogenic in the rat it does, however, appear to depress the rate of body weight gain in the pups at 4% in the diet. The number of live born pups , and related to this Fertility Index (FI), Viability Index (VI) and Lactation Index (LI) also decreased at 4%. There were no histological effects on the gonads. NOEL for teratogenicity is 3000 mg/kg (4% in the diet); NOAEL for embryotoxicity is 750 mg/kg (1% in the diet).
In conclusion, a combined reproduction-teratogenicity study with read across substance CAS No. 37294 -49 -8 did not indicate potential for developmental toxicity.
Teratogenicity testing
Further data on prenatal developmental toxicity were available from read across substance Docusate sodium (CAS No. 577-11-7). Justification for read across with the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13.
-A key study for prenatal developmental toxicity was performed in rats dosed from day 6-15 of gestation with read across substance Docusate sodium dosed at dietary dose levels of 1.0 and 2.0 % in the diet (Roell et al., 1976). The study was conducted according to OECD 414 guideline, and was considered to be reliable, adequate and relevant. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Toxic dietary levels of 2.0% Docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophthalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophthalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. Interpretation of the results of the present experiment, in which only maternally toxic doses induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants. The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with a test article intake of 1074 mg/kg body weight, as calculated in the study.
- As supporting information, prenatal developmental toxicity was also studied in rats by dietary administration of Docusate 'calcium' (DCS) at dose levels of 0.5, 1.0, 1.5 and 2.0 % in the diet as well as by oral gavage at 250, 500, 750 and 1000 mg/kg bw (Roell et all., 1976). Subtoxic dietary levels of 0.5 and 1.0% Docusate calcium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 1.5 and 2.0% DCS produced significant incidences of resorptions and gross abnormalities consisting primarily of exencephaly of varying degrees with spina bifida, anophthalmia and associated skeletal defects. However, dietary levels of 2% of DCS fed to pregnant rats for 3 days (days 6-8, 8-10 or 10-12) did not produce teratogenic response. Also, DCS given to pregnant rats by oral intubation at maternally subtoxic doses (250-750 mg/kg) and a slightly toxic dose (1000 mg/kg) did not lead to malformations, however the incidence of resorptions was increased at the 2 toxic doses. Likewise doses of 500 and 750 mg/kg given by gavage from day 6-15 produced an increase in resorptions at the highest dose without a teratogenic effect. Since only maternally toxic doses fed on gestational day 6-15 produced embryotoxic and teratogenic effects, it is concluded that no real hazard exists.
In conclusion, both a prenatal developmental toxicity with read across substance Docusate sodium and with read across substance Docusate calcium were negative for teratogenicity at non-toxic dose levels.
Conclusion
An oral gavage reproductive screening study with read across substance Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268-36-3) showed NOAEL of 60 mg/kg bw for paternal/maternal systemic toxicity, whereas 120 mg/kg bw was NOAEL for reproductive and developmental toxicity. The latter was based on reduced gestation index, (life) birth index and No. of pups and increased post-implantation loss at 300 mg/kg bw.
A combined reproduction-teratogenicity study with read across test item Disodium C-isodecyl sulphonatosuccinate (CAS No. 37294-49-8) in 2 generations resulted in NOAEL for embryotoxicity of 750 mg/kg (1% in the diet) and a NOEL for teratogenicity of 3000 mg/kg (4% in the diet).
Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium (CAS 577-11-7) in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw, whereas at 2% in the diet visceral and skeletal anomalies were observed, which was secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed.
Justification for classification or non-classification
Based on these results and according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.
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