Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Existing data on reproductive toxicity as well as extended histopathology including specific investigation of reproductive organs in a 90 day oral (dietary) repeated dose toxicity study do not indicate specific concern for reproductive function (details see section 5.9.1. of CSR and IUCLID section 7.8.1 and section 5.6.1 of CSR and IUCLID section 7.5.1). Based on the available data, neither in the OECD 422 nor in the OECD 408 study specific substance related effects on reproduction performance are deducible. No signs or indications of reproductive toxicity were also revealed up to the highest dose of 60 mg/kg bw/day (OECD 422) or 75 mg/kg bw/day (OECD 408). The lowest available dose without effects on reproductive function therefore is 60 mg/kg bw/day which is considered the NO(A)EL for this endpoint.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2009 to 07 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
-strain: Wistar Crl:WI
-Source: Charles River, 97633 Sulzfeld, Germany
-The animals were derived from a controlled full barrier maintained breeding system (SPF)
-Age of the animals at the beginning of the study was 8-9 weeks. The range of the body weight was:
Females: 175 to 203 g, (mean: 184.95 g, ± 20%= 36.99 g)
Males: 257 to 290 g, (mean: 272.63 g, ± 20%= 54.53 g)
-Housing-The animals were kept individually in IVC cages, type III H, polysul-phone cages on Altromin saw fibre bedding
-Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
-Water (ad libitum): tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, muncipal residue control, microbiol. controlled periodically)
-Acclimation period 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 3 °C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour

IN-LIFE DATES: from 15 December 2009 to 07 February 2010
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
-Amount of vehicle: 10 mL/kg bw
-Concentration in vehicle: 1.5, 3.0, 6 mg/mL
Details on mating procedure:
mating ration 1:1, mating period- 14 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analyzed for nominal concentration. Homogeneity of the test item in the vehicle was analyzed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1
(First week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.
The dose formulation analysis was performed at BSL BIOSERVICE Scientific Laboratories GmbH.
The analytical report (BSL Study 093602) is added in Annexure 4.
Duration of treatment / exposure:
males: 28-29 days; females: Approx 54 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 15, 30, 60 mg/kg body weight
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were dosed with the test item on 7 days per week basis. The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.
The test item was administered by gavage using a gavaging cannula. The maximum dose volume administered was 10 mL / kg body weight.
Positive control:
no
Parental animals: Observations and examinations:
Cage Side Observations:
-Time Schedule: Along with clinical observations
-Cage side observation included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalization, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour

Detailed Clinical Observations:
-Time schedule: General Clinical observation were made twice a day

FOB:Sensory reactivity to different modalities, grip strength and motor activity assessments and other behaviour observations were conducted on five randomly selected males and females from each group. Observation was made during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated).

Body weight:

-weighed at randomisation; males weekly during the entire study period and at terminal sacrifice; females were weighed weekly during pre mating period, on gestation day 0, 7, 14, 20 and on PND 1 (within 24 hours of parturition) and 4 along with pups.

Food consumption:

-measured on corresponding day of body weight (in males only during pre mating period) after beginning of the dose administration. Food consumption was not measured during mating period.

Litter Observations:
-The duration of gestation was recorded and is calculated from day 0 of pregnancy.
-Each litter examined soon after delivery of the dam for the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities
-Live pups counted, sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum

Hematology:

-Time schedule for the collection of blood: day of necropsy
-Anaesthetic used for the blood collection: yes
-Animals fasted- no
-Parameters: Haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count,
platelet count, blood clotting time , and differential leucocytes count (On five randomly selected males and females)

Clinical Biochemistry:

-Time schedule for collection of blood: day of necropsy
-animals fasted: no
-Parameters: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, two enzymes indicative of hepatocellular effects ( alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase)- on five randomly selected males and females.

Urinalysis:

-Time Schedule for the collection of urine: day of necropsy- samples collected from five randomly selected males at the terminal sacrifice.

Sacrifice and pathology

Gross Pathology-
-Males were sacrificed after the completion of mating period (total dosing of 28-29 days)
-females were sacrificed on respective post natal day 4 along with pups by using high dose of sodium pentobarbital.
-In the animals randomly selected for the blood collection, anaesthesia solution (Ketamin/Xylazin, 2:1) was used. At the time of sacrifice or death during the study, the adult animals were subjected to a full, detailed gross necropsy which includeed careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents with special attention was paid to the organs of the reproductive system.
-The number of implantation sites and corpora lutea recorded for each parental female at necropsy
-Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter were carefully examined for gross abnormalities
-The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved
-Organ weight: Reproductive organs from all animals were weighed (testes, epididymides, prostate, seminal vesicle with coagulating glands as whole, ovaries, uterus with cervix as applicable). From five adult males and females randomly selected from each group, the wet weight of the liver, kidneys, adrenals, thymus, spleen, brain and heart were taken.

Histopathology:

Full histopathology was carried on the preserved organs and tissues of same five randomly selected animals in control and high dose group. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals. In addition, the evaluation of stomach (non glandular and glandular) and trachea were extended for LD and MD groups due to findings in HD group animals.
The organ list were as follows;
- all gross lesions
- brain (representative regions including cerebrum, cerebellum and pons)
- spinal cord
- mammary glands (female only)
- liver
- kidneys
- adrenals
- stomach
- small and large intestines (including Peyer´s patches)
- thymus
- thyroid
- spleen
- trachea and lungs
- heart
- urinary bladder
- lymph nodes (one lymph node covering the route of administration (mesenteric) and another one distant from the route of administration to cover systemic effects (axillar)
- peripheral nerve (e.g. N. ischiadicus/ N. tibialis) in close proximity to muscle
- section of bone marrow (sternum)


Histopathological processing was performed at Propath UK Ltd, Willow Court, Netherwood Road, GB - Hereford HR2 6JU.
Histopathological evaluation performed at GLP-certified test site KALEIDIS –Consultancy in Histopathology, 6 rue du Gers, 68300 Saint-Louis, France.




Estrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
-Each litter to establish the number of pups, sexof pups, stillbirths, live births, runts and for the gross abnormalities.
-Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.
- In addition to the observations on parent animals abnormal behaviour of the offspring, if any, was recorded.
Postmortem examinations (parental animals):
-hematology on blood samples
-clinical biochemistry
-urinalysis
-Macroscopic examination
-organ weight
-histopathology
Postmortem examinations (offspring):
-gross external examination
-full necropsy of abnormal/dead pups
Statistics:
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett‟s multiple comparison test was carried out to reveal any differences between control- and test groups. These statistics were performed with GraphPad Prism V.5 software (p<0.05 was considered as statistical significant).
Reproductive indices:
copulation index, fertility index, delivery index and viability index were calculated based on the data generated
Offspring viability indices:
between PND 0 and PND 4
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The clinical signs observed in female control and treatment groups are as follows.
Control- nibbling of fur;
LD, MD and HD- respiratory sound, salivation;
MD and HD- moving the bedding, piloerection, vaginal secretion, nibbling of fur, regurgitation, sneezing;
HD- weight loss, dyspnoea, cyanosis, coughing.
The incidences of clinical signs observed were higher in MD and HD groups of both sexes and could be attributed to toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item related mortalities and effect on functional and behavioural endpoints were observed in male and female animals during the entire study period. Three animals (1 female and two males) died prematurely in the study, which was due to gavaging error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant difference was observed for body weight development in male HD group during premating days 1-7 and premating day 1 to terminal sacrifice [1- TS (day 29/30)] and female MD and HD group during GD 14-20 compared to corresponding controls. The statistical deviation observed in Male HD group and female MD and HD groups may be attributable to the observed reduced food intake during the same period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the high dose of 60 mg/kg bw/day as well as in one dam of the mid dose with 30 mg/kg bw/day local irritating effects at the forestomach were observed. These findings are considered a direct consequence of the corrosive property of the test substance. Findings in the trachea are considered to be secondary effects due to reflux (regurgitation) and subsequent aspiration of gavaged test substance with following repetitive direct irritation of the nonglandular mucosa of the stomach by the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY

All animals survived except one female from the high-dose group (Animal no. 35) and two males from the high-dose group (Animal no. 73 and 79) till the end of the study.

No clinical signs were observed in control and LD groups of males. The following clinical signs were observed in MD and HD group animals.
MD- salivation, moving the bedding post application, piloerection, respiratory sound, alopecia; HD- salivation, moving the bedding post application, piloerection, respiratory sound, half eyelid closure, regurgitation, bloody nose. The clinical signs observed in female control and treatment groups are as follows. Control- nibbling of fur; LD- respiratory sound, salivation; MD- respiratory sound, salivation, moving the bedding, piloerection, vaginal secretion, nibbling of fur, regurgitation, sneezing; HD- respiratory sound, salivation, moving the bedding, piloerection, weight loss, dyspnoea, cyanosis, coughing. The incidences of clinical signs observed were higher in MD and HD groups of both sexes and could be attributed to toxicity.
Three animals (1 female and two males) died prematurely in the study, which was due to gavaging error.

NEUROBEHAVIOUR

No relevant differences were observed in males and females.

HEMATOLOGY
Except for RBC (male HD group), MCHC (Female MD group), PTT and aPTT (female LD group) no statistical significant deviation was recorded in any of the hematological parameters between the treatment group and the corresponding control values. The statistical deviation observed for MCHC (Female MD) and RBC (Male HD) may not be attributed to toxicity due to lack of dose response effect.


CLINICAL BIOCHEMISTRY

Except for SGPT values in male HD group no deviation was recorded in any of the parameters. This deviation recorded in SGPT value could be due individual incidental value and may not be attributed to the treatment as no microscopic findings were recorded in liver that could support this finding.

URINALYSIS

no effects

ORGAN WEIGHT

In males, except for absolute seminal vesicle weight in HD group, statistically no significant differences in the absolute and relative organ weights of the treatment groups was observed when compared with the controls. The deviation observed in absolute seminal vescicle weight cannot be attributed to toxicity as there was no such deviation recorded for relative seminal vesicle weight.
In females, statistically no significant differences in the absolute and relative organ weights of the treatment groups was observed when compared with the controls.

GROSS PATHOLOGY

At terminal sacrifice, macroscopic organ findings noted were mainly in MD nad HD group animals.

In males, the macroscopic findings observed were multiple white spots on mucosa of cardis (animals 66, 72, 74, 75 and 78); lung filled with foamy and bloody content (animals 73 and 79); white and thickening at mucosa cardia (animal 76, 80).
In females, macroscopic findings observed were discoloured lung with multiple spots (animals 21 and 27); multiple white/yellowish/greenish spots on mucosa of cardia (animals 31, 32, 33, 34, 37, 39 and 40); lung ,trachea, mouth and nose with bloody foam (animal 31) .

HISTOPATHOLOGY

In the female and male reproductive organs, no macroscopic or histopathological lesions considered to be test item-related were observed.

In the nonglandular part of the stomach, test item-related histopathological changes were noted in all animals evaluated from the HD group and in single animals evaluated from the MD group and comprised a multifocal/diffuse epithelial hyperplasia, mostly in combination with epithelial hyperkeratosis and, in a proportion of animals, with submucosal inflammatory edema.

In the trachea, a minor epithelial alteration/hyperplasia was seen in a small proportion of animals of the MD and HD groups.

REPRODUCTIVE PARAMETER

No significant difference was observed on precoital interval when compared with their corresponding controls. All pregnancies resulted in normal births. Successful mating resulted in 10/10 pregnancies in the control and LD groups and 9/10 pregnancies in MD and 8/9 in HD groups.

Slightly but not significant reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in HD (88.9 %) and MD groups (90%) as compared to Control (100%) and LD groups (100%).

No significant differences observed for any of the reproductive indices (copulation index, fertility index and delivery index and viability index) between the treatment and control groups.


Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity, no major toxicological findings
Remarks on result:
other:
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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
Histopathological findings:
not examined
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
not examined
PRE and POST NATAL DATA

Group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0, percent pre implantation loss and post implantation loss remained unaffected due to treatment when compared with controls.

LITTER DATA

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4.

PUP SURVIVAL

Survival of the pups from PND 0 to PND 4 remained unaffected due to treatment in all treatment groups.
At necropsy, no gross external abnormalities considered to be treatment related.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
gross pathology
other: Number of live pups born on PND 0, number of males and females, sex ratio uneffected compared to controls. Survival of pups from PND 0 to PND 4 remained unaffected in all treatment groups. No gross pathological findings observed.
Reproductive effects observed:
no
Conclusions:
In conclusion, in this combined repeated dose toxicity and reproduction/ developmental toxicity screening test with HYEQS, the repeated dose administration of HYEQS in deionised water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 15, 30 and 60 mg/kg body weight revealed no major toxicological findings.

Parameters like neurology, hematology, clinical biochemistry, organ weight and fertility in males and females remained unaffected. The deviation observed for body weight development and food consumption were not attributed to treatment. The prenatal and litter data also did not show any treatment related effect. However, the clinical/macroscopic findings observed in males/ females of MD and HD groups correlated with the microscopic findings (e.g. multifocal/diffuse epithelial hyperplasia in the nonglandular part of the stomach, mostly in combination with epithelial hyperkeratosis and with submucosal inflammatory edema and a minor epithelial alteration/hyperplasia in the trachea as noted in some animals). These findings are based on local effects due to the corrosive properties of the test substance.
Based on the data generated, the dosage 15 mg/kg/day is determined as No-Observed- Adverse-Effect-Level (NOAEL) for overall toxicity due to local histopathological findings in stomach and trachea of the parental animals. However, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity is believed to be 60 mg/kg /day in males and females
Executive summary:

Material and Methods

In this Combined Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test performed according to First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 422, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” adopted on 22 March 1996 in wistar rats with HYEQS suspended in deionised water as vehicle, orally administered in graduated doses to three groups of male and female rats (Wistar Crl:WI) by gavage using feeding tube. The main study included 4 groups (C, LD, MD and HD) with each 10 males and 10 females

The animals were dosed with the test item on 7 days per week basis. The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.

The test item was administered by gavage using a gavaging cannula. The maximum dose volume administered was 10 mL / kg body weight.

The following doses were chosen,

Low Dose: 15 mg/kg body weight

Mid Dose: 30 mg/kg body weight

High Dose: 60 mg/kg body weight

RESULTS

All animals survived except one female (Animal no. 35) and two males (Animal no. 73 and 79) till the end of the study. Male animals were sacrificed on day 29 and 30. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactating females along with pups were sacrificed on respective post natal day 4.

No test item related mortalities and effect on functional and behavioural endpoints were observed in male and female animals during the entire study period.

Statistically significant difference was observed for body weight development in male HD group during premating days 1-7 and premating day 1 to terminal sacrifice [1- TS (day 29/30)] and female MD and HD group during GD 14-20 compared to corresponding controls.

Statistically significant difference was observed for food consumption in male HD group during premating days 1-7.

Statistical analysis of reproduction and litter data revealed no treatment related effect on group mean litter weight, number of males, number of females, total litter weight, female litter weights on PND 0 and PND 4, percent pre implantation loss, post implantation loss, total number of pups born, sex ratio, live pups, still birth and runt on PND 0, total No. of live pups and sex ratio on PND 0 and 4, precoital interval, No. of corpora lutea, No. of implantation sites when compared with controls.

Statistical significant difference was observed for male litter weight on PND 0 and 4 in LD group, which had no toxicological relevance (as there was no dose dependence response recorded).

Survival of the pups from PND 0 to PND 4 remained unaffected due to treatment in all treatment groups.

In male and female, statistical significant effect was observed for RBC (male HD), MCHC (female MD), PTT (female LD) and aPTT (female LD) compared to their corresponding control, which had no toxicological relevance.

Urinalysis in five randomly selected males from each group revealed no test item related effect.

Statisitcal analysis of clinical biochemistry data revealed increase in SGPT value in male HD group compared to control. This finding may be attributed to individual incidental value.

Treatment related gross pathological findings were observed in the nonglandular part of the stomach in all animals evaluated from the HD group and in single animals evaluated from the MD group. The histopathological changes comprised of multifocal/diffuse epithelial hyperplasia, mostly in combination with epithelial hyperkeratosis and, in a proportion of animals, with submucosal inflammatory edema.

No test item related changes were observed in pups at necropsy or death during the study.

In males, statistically no significant differences in the absolute (except absolute seminal vesicle weight in male HD group) and relative organ weights of the treatment groups was observed when compared with the controls. The deviation of seminal vescicle weight was regarded as incidental.

The histopathological evaluation of male and female reproductive organs did not reveal any histopathological lesions considered to be treatment related.

CONCLUSION

In conclusion, in this combined repeated dose toxicity and reproduction/developmental toxicity screeing test with HYEQS, the repeated dose administration of HYEQS in deionised water to the male (28 -29 days) and female (Maximum 54 days) Wistar rats at dosages of 15, 30 and 60 mg/kg body weight weight revealed no major toxicological findings.

Parameters like neurology, hematology, clinical biochemistry, organ weight and fertility in males and females remained unaffected. The deviation observed for body weight development and food consumption were not attributed to treatment. The prenatal and litter data also did not show any treatment related effect.

However, the clinical/macroscopic findings observed in males/females of MD and HD groups correlated with the microscopic findings (e.g. multifocal/diffuse epithelial hyperplasia in the nonglandular part of the stomach, mostly in combination with epithelial hyperkeratosis and with submucosal inflammatory edema and a minor epithelial alteration/hyperplasia in the trachea as noted in some animals). These findings are based on local effects due to the corrosive properties of the test substance.

Based on the data generated, the dosage 15 mg/kg/day is determined as No-Observed_Adverse-Effect-Level (NOAEL) for overall toxicity due to local histopathological findings in stomach and trachea of the parental animals. However, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity is believed to be 60 mg/kg/day in males and females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
scientific well performed OECD 422 study, GLP conform
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Based on the available data, neither in the OECD 422 nor in the OECD 408 study specific substance related effects on reproduction performance are deducible. No signs or indications of reproductive toxicity were also revealed up to the highest dose of 60 mg/kg bw/day (OECD 422) or 75 mg/kg bw/day (OECD 408). No reproductive toxic effects at all were observed at the high dose treatment with 60 mg/kg bw/day which therefore is considered a NOAEL (fertility) and is used for the CSA.

Effects on developmental toxicity

Description of key information

A prenatal developmental study in Wistar rats (OECD 414) is provided in 2017. Pregnant females (24/dose group) were dosed by oral gavage at 0, 20, 40, 80 and 120 mg/kg bw /d HYEQS (solved in water as vehicle) beginning at day 6 of pregnancy up to day 19 of pregnancy. On day 20 all females were sacrificed by caesarian section.

Dullness, diarrhoea, piloerection and epistaxis (two females) were observed in 24 out of 24 female animals treated at 120 mg/kg body weight (high dose). All animals of the high dose group died during day 8 to day 16 of gestation (i.e. between day 4 and day 12 of treatment). Gross pathological local findings at stomach, forestomach and glandular stomach are considered a direct consequence of the chemical property of the test substance, namely being a cationic surfactant. These findings are:

-       Stomach – distended, moderately to markedly;

-       Fore stomach raised whitish foci, moderately to markedly;

-       Ulceration moderately to markedly;

-       Mucous membrane peeled off in some females;

-       Glandular stomach – reddened, slightly to moderately.

No mortality or clinical signs of toxicity were observed in all animals dosed up to 80 mg/kg bw/d. The evaluation of the reproductive organs of all the dams, pre and post implantation loss, gravid uterine weight, litter size, viability of the fetuses, fetus weight per litter, fetus weight, sex ratio of the fetuses and the external, visceral and skeletal examination of the fetuses revealed that HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the dose of 80 mg/kg body weight. Hence, under the present experimental conditions, HYEQS is not a teratogen.

In addition HYEQS was tested for potential reproductive parameter in an OECD 422 screening study. Additionally a guideline conform OECD 414 study is available for the chemically closely related analogue compound C8 -C10 Dimethyl hydroxyethyl ammonium chloride, which is used for read across purposes to support the assessment (For Read across justification refer to IUCLID section 13, Assessment reports). The use of this substance data as surrogate is justified as it is seen as a worst case, i.e. based on the shorter chain length, its lower molecular weight and thus its higher chemical reactivity.

Based on actual derived results from a prenatal developmental study in Wistar rats (OECD 414) performed with HYEQS in 2016 the NOAEL of 80 mg/kg bw/day is considered with regard to developmental effects.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
other: weight of evidence
Adequacy of study:
supporting study
Study period:
N/A
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted according to OECD Test Guideline 414 in conformance with GLPs. The study was well documented and conducted. However, feed consumption was not recorded on presumed day 3 of gestation, as recommended in the study plan, but was recorded at all other recommended time points. For Read across justification refer to IUCLID section 13, Assessment reports.
Justification for type of information:
Refer to Read across justification section 13 of IUCLID
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See rationality for reliability above.
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Crl:CD(SD)IGS BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Age at study initiation: 66 days at arrival at study laboratory
- Weight at study initiation: 218-254 g on day of group assignment
- Fasting period before study: no data
- Housing: individually, based on computer-generated random units
- Diet: ad libitum
- Water: chlorinated, ad libitum
- Acclimation period: at least 6 days

BREEDER MALE RATS:
Number of rats: 150
Approximate age at arrival: 71 days
Weight on the day after arrival: 245-337 g
Weight at cohabitation: 491-682 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): at least 10 (100% fresh air passed through 99.97% HEPA filters)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: no data To: no data
Route of administration:
oral: gavage
Vehicle:
other: reverse osmosis membrane processed deionized water (R.O. deionized water)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared at least once weekly at the Testing Facility. Prepared formulations were stored at room temperature, protected from light. Formulations were stirred continuously (magnetic stir plate with stir bar). Duplicate samples (10 mL) were taken from each concentration level on the first day of preparation and on the last day of preparation. Prior to shipment, 0.28 mL of a 37% formaldehyde solution was added to each sample.

DIET PREPARATION
- Type of food: Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO)
- Storage temperature of food: no data
- Feed analysis: Analyses were routinely performed by the feed supplier. No contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were detected by these analyses.

VEHICLE
- Concentration in vehicle: 0, 2.5, 5.0 and 10.0 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected on the first and last day of dosing for analysis.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 4 days
- Replacement of first male by another male with proven fertility if unsuccessful pairing: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: presence of spermatozoa in a smear of the vaginal contents and/or copulatory plug observed in situ plug referred to as GD 0
- Any other deviations from standard protocol: N/A
Duration of treatment / exposure:
Gestation days (GD) 6-19
Frequency of treatment:
Once daily
Duration of test:
Gestation days (GD) 6-19
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a dose-ranging study performed in the study laboratory
- Rationale for animal assignment: computer-generated randomization procedures based on body weight recorded on GD 0
- Other: N/A
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice each day
- Cage side observations included: checks for viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly during the acclimation period and on GD 0
- Additional observations: clinical signs of effects of the test substance, abortions, premature deliveries and deaths, performed before dosing, approximately 60 ± 10 min after dosing administration and on the day of sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the acclimation period, on GD 0, daily during the dosing period and at sacrifice

FOOD CONSUMPTION: 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: N/A
- Time schedule: GD 0, 6, 9, 12, 15, 18 and 20

WATER CONSUMPTION: No data
- Time schedule for examinations: N/A

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: 20
- Organs examined: thoracic, abdominal and pelvic viscera, tissues with gross lesions

OTHER: Rats that were found dead were examined for the cause of death or moribund condition on the day the observation was made. The rats were examined for gross lesions.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The uteri of apparently non-pregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites.

Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter (soft tissue group)

Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.

Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variance and the Analysis of Variance, when appropriate [i.e., Bartlett's Test was not significant (p> 0.001)]. If the Analysis of Variance was significant (p≤ 0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p≤ 0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤ 0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data.

Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
N/A
Historical control data:
N/A
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY:
Three female rats in the 100 mg/kg/day dosage group were found dead. These deaths were attributed to effects of the test substance because they occurred at the highest dosage tested and the incidence was significantly increased (p≤ 0.01). All other rats survived until scheduled sacrifice on day 20 of gestation (GD 20).

The first dam (in the 100 mg/kg/day dosage group) was found dead on GD 19, 36 minutes after the 14th daily dosage. Adverse clinical observations included soft or liquid feces on GDs 16 to 18, excessive salivation on GDs 17 to 18 and chromorhinorrhea, urine-stained abdominal fur and ungroomed coat on GDs 18 to 19. This dam lost body weight and feed consumption values were reduced after GD 15. No gross lesions were revealed by necropsy. The litter consisted of 12 fetuses. All fetuses appeared normal at gross external and soft tissue examinations. One fetus had not ossified pubes and 1st sternal center, two fetuses had not ossified pubes and another fetus had not ossified 1st sternal center; all other fetuses examined for skeletal alterations appeared normal.

The second dam (in the 100 mg/kg/day dosage group) was found dead on GD 18 after 12 daily dosages. Adverse clinical observations included chromorhinorrhea, excessive salivation, urine-stained abdominal fur and soft or liquid feces on GDs 16 to 17. This dam lost body weight after GD 13 and had reduced feed consumption values on GDs 12 to 15. No gross lesions were revealed by necropsy. There were 12 late and 1 early resorptions in utero. Autolysis precluded examination of conceptuses for gross external alterations and sex.

The third dam (in the 100 mg/kg/day dosage group) was found dead on GD 20 after 14 daily dosages. Adverse clinical observations included urine-stained abdominal fur on GDs 15 to 19, soft or liquid feces on GDs 16 to 18, chromorhinorrhea and excessive salivation on GDs 16 to 19 and ungroomed coat on GDs 18 to 19. This dam generally lost body weight after GD 11 and reduced feed consumption values after GD 9. No gross lesions were revealed by necropsy. The litter consisted of 13 fetuses. All fetuses appeared normal at gross external and soft tissue examinations. One fetus had not ossified pubes; all other fetuses examined for skeletal alterations appeared normal.

MATERNAL BODY WEIGHTS AND BODY WEIGHT CHANGES:
Maternal body weight gains were reduced at all tabulated intervals during the dosage period in the 100 mg/kg/day dosage group; the reduction was significant (p≤ 0.01) on GDs 9-12. As a result of these reductions, maternal body weight gain was significantly reduced (p≤ 0.01) in the 100 mg/kg/day dosage group for the entire dosage period (calculated as GDs 6-20). Maternal body weight gains in the 100 mg/kg/day dosage group were also significantly reduced (p≤ 0.05 or p≤ 0.0 1) for the entire dosage and gestation periods when calculated using the corrected GD 20 body weight (GD 20 body weight minus the gravid uterine weight). Maternal body weights were significantly reduced (p≤ 0.05 or p≤ 0.01) in the 100 mg/kg/day dosage group on GDs 14 through 18. The GD 20 body weight corrected for the gravid uterine weight (GD 20C) was also significantly reduced (p≤ 0.05) in the 100 mg/kg/day dosage group.

MATERNAL ABSOLUTE AND RELATIVE FEED CONSUMPTION VALUES:
Absolute (g/day) and relative (g/kg/day) feed consumption values were reduced at all tabulated intervals during the dosage period in the 100 mg/kg/day dosage group; these reductions were significant (p≤ 0.01) on GDs 6-9 (relative only), 9-12 and 12-15. As a result of these reductions, absolute and relative feed consumption values were significantly reduced (p≤ 0.05 or p≤ 0.01) in the 100 mg/kg/day dosage group for the entire dosage period (calculated as GDs 6-20) and for the entire gestation period (GDs 0-20).

Absolute and relative feed consumption values were unaffected by dosages of the test substance as high as 50 mg/kg/day.

CLINICAL OBSERVATIONS:
The incidences of excess salivation, soft or liquid feces, urine-stained abdominal fur, chromorhinorrhea and ungroomed coat were significantly increased (p≤ 0.01) in the 100 mg/kg/day dosage group, as compared to the vehicle control group. These observations tended to first occur during the third week of gestation (GD 15-18) and were considered test substance-related. All other clinical observations were considered unrelated to the test substance because the incidences were not dosage-dependent or the observation occurred in only one rat. These observations included localized alopecia of the limbs, limited use of and swollen left hindlimb and paw, hunched posture and chromodacryorrhea.

NECROSPY OBSERVATIONS:
The only necropsy observations were slight dilation of the renal pelvis in one 100 mg/kg/day dosage group rat and large adrenals and distended stomach in another 100 mg/kg/day dosage group rat. These observations were considered unrelated to the test substance because they occurred in single rats. No other gross lesions were revealed by necropsy.

CAESAREAN SECTIONING OBSERVATIONS:
There were 22 (88%), 24 (96%), 24 (96%) and 24 (96%) pregnant rats in the 0 (vehicle), 25, 50 and 100 mg/kg/day dosage groups, respectively. As a result of the deaths in the 100 mg/kg/day dosage group, Caesarean-sectioning observations were based on 22, 24, 24 and 21 pregnant rats in the 0 (vehicle), 25, 50 and 100 mg/kg/day dosage groups, respectively. No other Caesarean-sectioning parameters were affected by dosages as high as 50 mg/kg/day. The litter averages for corpora lutea, implantations, early and late resorptions and percent dead or resorbed conceptuses were comparable among the four dosage groups and did not significantly differ. All placentas appeared normal.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: yes

Details on embryotoxic / teratogenic effects:
LITTER OBSERVATIONS:
Fetal body weights (total, male and female) were reduced in the 100 mg/kg/day dosage group. The average body weight of the male fetuses was significantly reduced (p≤ 0.05), compared to the vehicle control group value. All of these values were, however, within the ranges observed historically at the testing facility. One dam in the 100 mg/kg/day dosage group had a litter consisting of 15 dead fetuses. No other litter parameters were affected by dosages as high as 50 mg/kg/day. The litter averages for litter sizes, live fetuses and percent live male fetuses were comparable among the four dosage groups and did not significantly differ.

FETAL ALTERATIONS:
Fetal evaluations were based on 307, 343, 344 and 290, GD 20 Caesarean-delivered live fetuses in 22, 24, 24 and 20 litters in the 0 (vehicle), 25, 50 and 100 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 150, 165, 165 and 140 fetuses were examined for soft tissue alterations, and 157, 178, 179 and 150 fetuses were examined for skeletal alterations and fetal ossification site averages.

It was possible to examine the 15 dead fetuses from the one litter in the 100 mg/kg/day dosage group. All fetuses appeared normal of gross external and soft tissue examinations. Of the eight fetuses examined for skeletal alterations, all fetuses had not ossified pubes. One fetus also had a not ossified 1 sternal center and two fetuses also had not ossified ischium.

The litter and fetal incidences of not ossified 1st sternal centers and pubic bones were increased in the 100 mg/kg/day dosage groups; the fetal incidences of these observations were significantly increased (p≤ 0.01). These variations in skeletal development were considered related to the test substance and secondary to the fetal weight reductions that occurred in this dosage group.

No other gross external, soft tissue or skeletal alterations (malformations or variations) were related to treatment with the test substance as high as 100 mg/kg/day.

FETAL GROSS EXTERNAL ALTERATIONS:
No significant effects were reported for any test substance treatment group.

FETAL SOFT TISSUE ALTERATIONS:
The umbilical artery descended to the left of the urinary bladder in one fetus in the 25 mg/kg/day dosage group and in one fetus in the 50 mg/kg/day dosage group. No additional alterations occurred in these fetuses. The innominate artery was absent in one fetus in each of the 0 (vehicle) and 100 mg/kg/day dosage groups. No additional alterations occurred in these fetuses.

FETAL SKELETAL ALTERATIONS:
A bifid centrum in a thoracic vertebra occurred in one fetus in the 50 mg/kg/day dosage group and in two fetuses in the 100 mg/kg/day dosage group. No additional alterations occurred in these fetuses. A cervical rib at the 7th cervical vertebra, a common variation in this strain of rat, was present as the only alteration in one fetus in the 50 mg/kg/day dosage group.

Delayed sternal ossification (incompletely ossified and/or not ossified 1st and/or 2nd sternebrae), a reversible delay, occurred in two, one, four and nine (p≤ 0.01) fetuses from two, one, three and four litters from the 0 (vehicle), 25, 50 and 100 mg/kg/day dosage groups, respectively. Three fetuses in the 100 mg/kg/day also had incomplete ossified pubes. No additional alterations occurred in the other fetuses. The number of fetuses with incompletely ossified sternal centers, a reversible delay in ossification, was significantly increased (p≤ 0.01) in the 100 mg/kg/day dosage group. This significant increase was associated with reduced fetal body weights in this dosage group and considered related to the test substance because it was dosage-dependent.

The ischia and/or pubes were incompletely or not ossified in 3, 6, 1 and 10 fetuses from 2, 4, 1 and 5 litters in the 0 (vehicle), 25, 50 and 100 mg/kg/day dosage groups. The number of fetuses with incompletely ossified pubes, a reversible delay in ossification, was significantly increased (p≤ 0.01) in the 100 mg/kg/day dosage group. The significant increase was associated with the reduced fetal body weights in this dosage group and considered related to the test substance because it was dosage-dependent.

No statistically significant or biologically important differences occurred among the four dosage groups in the average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar, sacral and caudal), ribs, sternum (manubrium, sternal centers and xiphoid), forelimbs (carpals, metacarpals and phalanges) or hindlimbs (tarsals, metatarsals and phalanges).

Key result
Dose descriptor:
NOAEL
Effect level:
<= 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Develomental
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

N/A

Conclusions:
On the basis of these data, the maternal no observable adverse effect level (NOAEL) of the test substance is 50 mg/kg/day (the 100 mg/kg/day dosage caused mortality, increased incidences of adverse clinical observations and reductions in body weight gain and feed consumption values). The developmental NOAEL is 50 mg/kg/day (the 100 mg/kg/day dosage caused a reduction in fetal body weights and delays in skeletal ossifications).
Executive summary:

One hundred presumed pregnant Crl:CD®(SD)IGS BR VAF/Plus® rats were randomly assigned to four dosage groups (Groups I through IV), 25 rats per group. Formulations of the test substance, SS0772.01, or the vehicle, R.O. Deionized Water, were administered orally via gavage once daily to these naturally-bred female rats on days 6 through 19 of presumed gestation (GDs 6-19) at dosages of 0 (vehicle), 25, 50 and 100 mg/kg/day. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance or vehicle.

Checks for viability were made twice daily. Clinical observations were recorded daily before dosage and approximately 60 minutes after administration. These observations were also recorded on the day of sacrifice (GD 20). Body weights were recorded daily during the dosage period and on the day of sacrifice (GD 20). Feed consumption values were recorded on GDs 0, 6, 9, 12, 15, 18 and 20.

All rats were sacrificed on GD 20 and examined for the number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The gravid uterus was excised and weighed. Fetuses were weighed and examined for gross external alterations and sex. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations by using a variation of the microdissection technique of Staples. The remaining fetuses were examined for skeletal alterations after staining with alizarin red S.

Three female rats in the 100 mg/kg/day dosage group were found dead (one rat on each of GDs 18, 19 and 20). The incidences of excess salivation, soft or liquid feces, urine-stained abdominal fur, chromorhinorrhea and ungroomed coat were significantly increased in the 100 mg/kg/day dosage group. These observations tended to first occur during the third week of gestation (GDs 15-18) and were considered test substance-related.

Maternal body weight gains were reduced at all tabulated intervals during the dosage period in the 100 mg/kg/day dosage group. As a result of these reductions, maternal body weight gain was significantly reduced in the 100 mg/kg/day dosage group for the entire dosage period. Maternal body weight gains in the 100 mg/kg/day dosage group were also significantly reduced for the entire dosage and gestation periods when calculated using the GD 20 body weight corrected for gravid uterine weight. Absolute and relative feed consumption values were reduced in the 100 mg/kg/day dosage group for the entire dosage period and for the entire gestation period. Fetal body weights were reduced in the 100 mg/kg/day dosage group. No other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 50 mg/kg/day.

The litter and fetal incidences of not ossified 1st sternal centers and pubic bones were increased in the 100 mg/kg/day dosage groups; the fetal incidences of these observations were significantly increased. These variations in skeletal development were considered related to the test substance and secondary to the fetal weight reductions that occurred in thie dosage group.

On the basis of these data, the maternal no observable adverse effect level (NOAEL) of the test substance is 50 mg/kg/day (the 100 mg/kg/day dosage caused mortality, increased incidences of adverse clinical observations and reductions in body weight gain and feed consumption values). The developmental NOAEL is 50 mg/kg/day (the 100 mg/kg/day dosage caused a reduction in fetal body weights and delays in skeletal ossifications).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-02 to 2017-02-21
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted by the Council on 22nd January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: ESD0021175
- Expiration date of the lot/batch: 2017-11-27
- Apperance: colourless waxy solid
- Storage condition of test material: Refrigerator (2 to 8 °C)
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Vehicle:
other: Distilled water
Details on exposure:
The dose formulations were prepared shortly before each dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dose formulations from one set per dose group will be taken immediately after preparation once before commencement of dosing i.e. on day 5 of gestation and on day 19 of gestation (i.e. day 1 and day 15 of treatment) or homogeneity (mean of homogeneity will be given as dose concentration). On day 11 of gestation (e.e. day 7 of treatment), samples of all dose formulations will be analysed for dose concentrations by analysing triplicate samples.
Analysis was performed according to the validated analytical method provided by the sponsor.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one female
- Length of cohabitation: maximum period of fourteen days
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: Each female examined for presence of vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
Duration of treatment / exposure:
day 5 of Gestation to day 19 of Gestation
Frequency of treatment:
daily
Duration of test:
Animals were sacrificed on day 20 of gestation by cesariean section.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
females: 24 / group
males: 50 males in total were used for mating
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results from a dose range finding study in Wistar rats with the test substance (RCC study number 6265), conducted at RCC Laboratories India Private Limited, the 4 doses were selected.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Once on day of receipt and twice daily thereafter. Once on day of necropsy
-
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Acclimatization period once daily. Twice daily on initial 3 days after treatment; once daily thereafter

BODY WEIGHT: Yes
Time schedule for examinations:
- Acclimatization Period: Once weekly
- Premating: On first day of pairing and weekly thereafter (if any)
- Gestation: On gestation day 0, 3, 5, 8, 11, 14, 17, 19 and 20 and at death, if any

FOOD CONSUMPTION GESTATION: Yes
- on Gestation day 3, 5, 8, 11, 14, 17 and 19

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
All animals including those found dead or sacrificed in moribund condition were subject of complete gross necropsy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead during study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per littera
- Skeletal examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the body weight, body weight change, feed consumption, reproduction and external, visceral and skeletal alterations/variations.
•Data were summarized in tabular form. Statistical analysis were performed using statplus program. Values were given as mean ± standard deviation (SD).
•All the data were checked for Normality with Shapiro-Wilk W test.
•All the data were checked for Homogeneity with Bartlett Chi-Square test.
•Data were subjected to perform Analysis of Variance (ANOVA) for continuous data.
•Discontinuous data were subjected to nonparametric test (Mann-Whitney U-Test).
Indices:
The following formulae were used for calculating the indices:
Pre–implantation loss (%)
(Number of Corpora Lutea - number of implantation sites) / Number of Corpora Lutea x 100
Post–implantation loss (%)
(Number of implantation sites - Total number of live foetuses) / Number of implantation sites x 100
Sex Ratio (% males)
Number of male foetuses (Day 0) / Total number of foetuses (Day 0) x 100
Variation Incidence (%)
Number of foetuses with variation/ Total Number of foetuses examined x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs observed in all females up to 80 mg/kg bw/d. Prior to death of all group 120 mg/kg bw/d animals, these animals showed diarrhoea, dullness and piloerection. Two animals additionally showed epistaxis.
Mortality:
mortality observed, treatment-related
Description (incidence):
No Mortalities observed up to the dose of 80 mg/kg bw/d. All females of 120 mg/kg bw dose group died beginning on treament day 4 until day 12 of treatment. Prior to death, all animals showed diarrhoea, dullness and piloerection. Two animals additionally showed epistaxis.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference was observed in the body weight and body weight gain (%) of pregnant females in low, intermediate and medium high dose group animals as compared with control group animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant difference was observed in the feed consumption of pregnant females in low, intermediate and medium high dose group animals when compared with control group animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopy did not reveal any abnormalities during last week in all animals of groups 1 and 4. In the last week of treatment, all the animals in group 5 were found dead. Hence, ophthalmoscopy was performed for group 4 animals.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters such as Erythrocyte count (RBC), Hemoglobin (Hb), Hematocrit (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Total leucocyte count (WBC), Differential leukocyte count (DC), Prothrombin time (PT) and Activated Partial Thromboplastin time (APTT) did not show any toxicologically relevant findings in the treated groups when compared with control group.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters like Glucose (GLU), Urea, Creatinine (CREA), Triglycerides (TRIGL), Alkaline phosphatase (ALP), Bilirubin (BIL), Sodium (Na), Potassium (K), Chloride (Cl), Total protein (TPO), Albumin (ALB), Globulin (GLB) and Albumin and Globulin ratio (A/G) did not show any toxicologically relevant findings in treatment groups when compared with control group. Where as in cholesterol, there was significant decrease in medium high as compared with intermediate dose. In aspartate aminotransferase, there was significant increase in medium high as compared with intermediate dose and significant decrease in intermediate dose as compared with low dose. In alanine aminotransferase, there was significant increase in low, intermediate and medium high dose as compared with control and significant increase in medium high as compared with low and intermediate dose.
The significant changes observed in the clinical pathology parameters were marginal and could not be attributed to the test item administration as these values were within clinical and biological variation.
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:
effects observed, treatment-related
Description (incidence and severity):
No test item-related macroscopic findings were observed at necropsy on day 20 of gestation in any of the animals up to 80 mg/kg bw/d. All Females of 120 mg/kg bw dose group died beginning on treatment day 4 until day 12 of treatment. Findings at necropsy of 120 mg/kg bw/d dose group females: Thymus - small in size, slightly; Adrenals – enlarged, slightly; Spleen – enlarged, slightly; Stomach – distended, moderately to markedly; Fore stomach raised whitish foci, moderately to markedly; Ulceration moderately to markedly; Mucous membrane peeled off in some females; Glandular stomach – reddened, slightly to moderately.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions occurred during the treatment period.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups. No litter loss occurred
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
With the exception of one dead fetus out of 170 fetuses at the 80 mg/kg bw/d dose group, no dead fetuses were observed in any of the dose groups (0, 20, 40, 80 mg/kg bw/d).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Due to the study design of an OECD 414 study, pregnancy is terminated by caesarian section at day 20 of gestation. No changes were observed at any dose group.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Due to the study design of an OECD 414 study, pregnancy is terminated by caesarian section at day 20 of gestation.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All female animals of control, low, intermediate, medium high and high dose groups showed positive evidence of mating. At necropsy, three females each in control and 40 mg/kg bw/d group, four females in 20 mg/kg bw/d dose group and two females in the 80 mg/kg bw/d dose group were found non pregnant. In high dose group (i.e. 120 mg/kg bw/d) three animals were found non pregnant during necropsy.
Other effects:
no effects observed
Details on maternal toxic effects:
All Females of 120 mg/kg bw dose group died beginning on treatment day 4 until day 12 of treatment. Findings at necropsy of 120 mg/kg bw/d dose group females: Thymus - small in size, slightly; Adrenals – enlarged, slightly; Spleen – enlarged, slightly; Stomach – distended, moderately to markedly; Fore stomach raised whitish foci, moderately to markedly; Ulceration moderately to markedly; Mucous membrane peeled off in some females; Glandular stomach – reddened, slightly to moderately. At necropsy no abnormality was detected in all females of dose groups 0, 20, 40, 80 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
Remarks on result:
other:
Remarks:
At high dose of 120 mg/kg bw/d all dams died
Key result
Abnormalities:
no effects observed
Localisation:
other: none
Fetal body weight changes:
no effects observed
Description (incidence and severity):
At time of ceasarian section and thus time point of weighing all foetuses, no significant difference was observed in foetal weight between control and treated groups. Foetal body wheight change is not an aim of the study design.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At time of ceasarian section which is part of the OECD 414 study design and thus time point of weighing all foetuses, no significant difference was observed in foetal weight between control and treated groups. Foetal body wheight change is not an aim of the study design.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No reduction of live foetuses was observed throughout the dose groups 0, 20, 40 and 80 mg/kg bw/d. The high dose group (i.e. 120 mg/kg bw/d) could not be evaluated as all dams of this dosing group died until treatment day 12.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was calculated as percent (%) male and no significant difference was observed in sex ratio in treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No changes in litter size and litter weights could be determined in all treated groups compared to control group.
Changes in postnatal survival:
not specified
Description (incidence and severity):
Postnatal survival is not an aim of the study design of an OECD 414 study.
External malformations:
no effects observed
Description (incidence and severity):
There were no test item related variations/malformations observed in foetuses subjected for external and visceral (soft tissue) examination.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of foetuses showing skeletal variation in treatment groups were comparable with control group. There were no test item related skeletal variations/malformations in any of the treated group. Most of the observations recorded were minor abnormalities and considered as routine variables.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test item related variations/malformations observed in foetuses subjected for external and visceral (soft tissue) examination.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
<= 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the medium high dose of 80 mg/kg bw/d.
Remarks on result:
other:
Remarks:
At high dose of 120 mg/kg bw/d all dams died
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Tab. 1            Mortality and Clinical Signs Females - Summary

 

Group

G1

G2

G3

G4

G5

Dose (mg/kg bw)

0

20

40

80

120

Mortality

0/24

0/24

0/24

0/24

24/24

Clinical signs

0/24

0/24

0/24

0/24

Dullness

-

(24/24)

Diarrhoea

-

(24/24)

Piloerection

-

(24/24)

Epistaxis

-

(02/24)

 

 

Tab. 2            Pregnancy Status - Summary

 

Group

G1

G2

G3

G4

G5*

Dose (mg/kg bw)

0

20

40

80

120

Females mated

24

24

24

24

24

Pregnant females

21

20

21

22

21

* All the animals were found dead between day 8 and 16 of gestation period (i.e. day 4 and day 12 of treatment)

 

 

Tab. 3            Body weight (G) dams - Summary

Group

Body weights (G)

Gestation Day

 

0

3

5

8

11

14

17

20

Group 1

Mean

253.6

261.8

269.0

276.5

288.0

303.2

319.1

329.4

SD

7.44

8.67

10.44

11.42

13.34

15.01

14.82

14.04

N

21

21

21

21

21

21

21

21

Group 2

Mean

255.8

261.7

267.7

274.7

283.8

297.0

313.5

327.1

SD

9.27

9.25

9.80

11.75

13.47

13.35

14.11

15.30

N

20

20

20

20

20

20

20

20

Group 3

Mean

256.8

262.6

268.5

275.1

286.2

302.3

322.6

338.4

SD

8.25

6.97

6.68

7.87

7.35

10.08

11.49

15.02

N

21

21

21

21

21

21

21

21

Group 4

Mean

265.0

262.5

269.7

277.3

287.8

301.0

319.1

333.4

SD

8.14

7.91

9.44

11.30

12.41

13.04

14.59

16.26

N

22

22

22

22

22

22

22

22

 

 

Tab. 4            Body weight Gain (%) dams - Summary

Group

Body weights Gain (%)

Gestation Day

-

0

3

5

8

11

14

17

20

Group 1

Mean

0.0

3.2

6.1

9.0

13.6

19.6

25.9

29.9

SD

0.00

1.91

3.38

4.31

4.78

5.44

5.52

5.38

N

21

21

21

21

21

21

21

21

Group 2

Mean

0.00

2.3

4.7

7.5

11.0

16.2

22.7

28.0

SD

0.00

1.47

2.66

3.90

4.52

4.56

5.93

7.10

N

20

20

20

20

20

20

20

20

Group 3

Mean

0.0

2.3

4.6

7.2

11.5

17.8

25.7

31.9

SD

0.00

1.30

2.54

3.90

3.92

5.21

6.42

7.73

N

21

21

21

21

21

21

21

21

Group 4

Mean

0.0

2.6

5.4

8.4

12.5

17.6

24.8

30.4

SD

0.00

1.77

3.18

4.51

5.07

5.51

7.15

8.75

N

22

22

22

22

22

22

22

22

 

 

Caesarean Data

Tab. 5            Maternal Data - Summary

 

Parameter

Group

G1

G2

G3

G4

Dose (mg/kg bw)

0

20

40

80

No. of Dams

21

20

21

22

Gravid Uterine Weight (G)

Mean

41.5978

38.2861

50.8140

41.9012

SD

19.0702

20.3954

15.4402

20.3170

Total Foetuses

Number

163

136

183

170

Mean

7.76

6.80

8.71

7.73

SD

3.83

4.09

3.15

4.08

No. of CL

Mean

12.48

11.60

12.14

11.05

SD

3.37

2.64

2.52

4.71

No. of Implantation

Mean

8.48

7.90

9.48

8.82

SD

3.92

4.36

3.03

4.26

Early Resorption

Mean

0.67

0.90

0.76

1.09

SD

0.86

1.12

1.22

1.31

Late Resorption

Mean

0.05

0.20

0.00

0.00

SD

0.22

0.89

0.00

0.00

Pre implantation loss

Mean

4.0

3.7

2.7

2.2

SD

3.44

3.34

3.26

2.20

%

30.85

34.23

20.75

18.38

Post implantation loss

Mean

0.7

1.1

0.8

1.1

SD

0.90

1.29

1.22

1.39

%

9.07

14.19

8.75

14.75

Total Number of resorption/s

Number

15

22

16

24

 

 

Pup Data

Tab 6             Litter data – Summary

 

Parameter

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Dams

24

24

24

24

No. of Litters

21

20

21

22

Total No. of Foetuses

163

136

183

170

Mean Litter Size

7.76

6.80

8.71

7.73

No. of live foetuses

Number

163

136

183

169

Mean

7.76

6.80

8.71

7.68

SD

3.83

4.09

3.15

4.09

(%)

100

100

100

99.4

No. of dead foetuses

Number

0

0

0

1

Mean

0.00

0.00

0.00

0.05

SD

0.00

0.00

0.00

0.21

No. of live male foetuses

Number

70

72

80

84

Mean

3.33

3.60

3.81

3.82

SD

1.85

2.48

1.91

2.56

No. of live female foetuses

Number

93

64

103

85

Mean

4.43

3.20

4.90

3.86

SD

2.60

1.91

2.26

2.53

 

 

Tab.7             Group Mean Foetal weight

 

Group

Foetal weight /

Litter (G)

Foetal weight (G)

Male

Female

Group 1

Mean

3.5834

3.6996

3.4960

SD

0.6132

0.6325

0.5866

N

163

70

93

Group 2

Mean

3.6514

3.7097

3.5859

SD

0.5052

0.5193

0.4845

N

136

72

64

Group 3

Mean

3.8994

4.0176

3.8076

SD

0.6315

0.5102

0.7004

N

183

80

103

Group 4

Mean

3.7112

3.7236

3.6990

SD

0.5006

0.4952

0.5085

N

170

84

86

 

 

Tab. 8            Group Mean Sex Ratio

 

Group

Sex ratio (%)

Male

Sex ratio (%)

Female

Group 1

Mean

41.75

58.25

SD

15.73

15.73

N

163

Group 2

Mean

47.02

52.98

SD

25.65

25.65

N

136

Group 3

Mean

41.50

58.50

SD

19.44

19.44

N

183

Group 4

Mean

44.38

51.31

SD

23.66

24.21

N

170

 

 

Effects on Embryos/Foetuses

Tab. 9            External findings - Summary

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Litter Examined

21

20

21

22

No. of Foetuses Examined

163

136

183

170

Variation Incidence – Number (%)

No. of Foetus with Variations

Total Variations

1(0.61)

1(0.74)

2(1.10)

1(0.59)

Body Small in size

1(0.61)

1(0.74)

1(0.55)

1(0.59)

Body Pale

0(0.00)

0(0.00)

1(0.55)

0(0.00)

 

 

Tab. 10           Visceral Findings - Summary

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Litter Examined

21

20

21

22

No. of Foetuses Examined

76

63

87

81

Variation Incidence – Number (%)

No. of Foetus with Variations

Total Variations

7(9.21)

1(1.59)

0(0.00)

2(2.47)

Ureters: Convoluted

4(5.26)

0(0.00)

0(0.00)

2(2.47)

Spleen: Pale

2(2.63)

0(0.00)

0(0.00)

0(0.00)

Spleen: Small in size

1(1.32)

1(1.59)

0(0.00)

0(0.00)

 

 

Tab. 11           Skeletal Findings - Summary

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

20

40

80

No. of Litter Examined

21

20

21

22

No. of Foetuses Examined

87

73

96

89

Variation Incidence – Number (%)

Nasal

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Premaxilla

Not Ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Mandible

Short

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Exoccipital

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Interparietal

Incompletely ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Supraoccipital

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Hyoid

Incompletely ossified

1(1.16)

0(0.00)

1(1.04)

0(0.00)

Vertebrae - Thoracic

Dumbbell shaped

7(8.14)

13(17.81)

14(14.58)

14(15.73)

Bipartite

2(2.33)

0(0.00)

2(2.08)

5(5.62)

Cervical

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Caudal

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Lumbar

Dumbbell shaped

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Hemivertebra

0(0.00)

1(1.37)

0(0.00)

0(0.00)

Misaligned

0(0.00)

1(1.37)

0(0.00)

0(0.00)

Sacral

Dumbbell shaped

0(0.00)

1(1.37)

0(0.00)

0(0.00)

Sternal Centers

Not ossified

9(10.47)

2(2.74)

2(2.08)

2(2.25)

Manubrium

Not ossified

0(0.00)

0(0.00)

1(1.04)

0(0.00)

Xyphiod

Not ossified

6(6.98)

2(2.74)

4(4.17)

1(1.12)

Ribs (Left)

Waviness

0(0.00)

0(0.00)

2(2.08)

3(3.37)

Supernumerary

12(13.95)

11(15.07)

14(14.58)

11(12.36)

Ribs (Right)

Waviness

0(0.00)

1(1.37)

2(2.08)

5(5.62)

Supernumerary

11(12.79)

11(15.07)

11(11.46)

12(13.48)

Ischium (Left)

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Ischium (Right)

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

Pubis (Left)

Not ossified

2(2.33)

0(0.00)

0(0.00)

0(0.00)

Pubis (Right)

Not ossified

1(1.16)

0(0.00)

0(0.00)

0(0.00)

 


 

 

Individual Macroscopic Findings Females

 

Group / Dose

Dam Number

Necropsy Findings

1/

0 mg/kg

 

01

No Abnormality Detected

02

No Abnormality Detected

03

No Abnormality Detected

04

No Abnormality Detected

05

No Abnormality Detected

06

No Abnormality Detected

07

No Abnormality Detected

08

No Abnormality Detected

09

No Abnormality Detected

10

No Abnormality Detected

11

No Abnormality Detected

12

No Abnormality Detected

13

No Abnormality Detected

14

No Abnormality Detected

15

No Abnormality Detected

16

No Abnormality Detected

17

No Abnormality Detected

18

No Abnormality Detected

19

No Abnormality Detected

20

No Abnormality Detected

21

No Abnormality Detected

22

No Abnormality Detected

23

No Abnormality Detected

24

No Abnormality Detected

Group / Dose

Dam Number

Necropsy Findings

2/

20 mg/kg

 

25

No Abnormality Detected

26

No Abnormality Detected

27

No Abnormality Detected

28

No Abnormality Detected

29

No Abnormality Detected

30

No Abnormality Detected

31

No Abnormality Detected

32

No Abnormality Detected

33

No Abnormality Detected

34

No Abnormality Detected

35

No Abnormality Detected

36

No Abnormality Detected

37

No Abnormality Detected

38

No Abnormality Detected

39

No Abnormality Detected

40

No Abnormality Detected

41

No Abnormality Detected

42

No Abnormality Detected

43

No Abnormality Detected

44

No Abnormality Detected

45

No Abnormality Detected

46

No Abnormality Detected

47

No Abnormality Detected

48

No Abnormality Detected

Group / Dose

Dam Number

Necropsy Findings

2/

20 mg/kg

 

25

No Abnormality Detected

26

No Abnormality Detected

27

No Abnormality Detected

28

No Abnormality Detected

29

No Abnormality Detected

30

No Abnormality Detected

31

No Abnormality Detected

32

No Abnormality Detected

33

No Abnormality Detected

34

No Abnormality Detected

35

No Abnormality Detected

36

No Abnormality Detected

37

No Abnormality Detected

38

No Abnormality Detected

39

No Abnormality Detected

40

No Abnormality Detected

41

No Abnormality Detected

42

No Abnormality Detected

43

No Abnormality Detected

44

No Abnormality Detected

45

No Abnormality Detected

46

No Abnormality Detected

47

No Abnormality Detected

48

No Abnormality Detected

 

Group / Dose

Dam Number

Necropsy Findings

 

5/

120 mg/kg

 

97

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Urinary bladder - distended, slightly;

Adrenals - enlarged, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci;

                 Ulcers, slightly;

Glandular stomach – reddened, slightly

 

98

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Urinary bladder - distended, slightly;

Adrenals - enlarged, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci  

                 moderately; Ulcers slightly;

Glandular stomach – reddened, slightly

 

99

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                 markedly; reddish fluid content present; Ulceration,

                 moderately;

Glandular stomach – reddened, slightly

 

100

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach - distended slightly; Fore stomach raised whitish foci

                 moderately; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

101

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Lungs – reddened, slightly;

Spleen - enlarged in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                 moderately; Ulceration, moderately;

Glandular stomach – reddened, moderately

 

5/

120 mg/kg

 

102

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size. slightly;

Stomach - distended slightly; Fore stomach raised whitish foci

                 markedly; reddish fluid content, present; Ulceration,

                 moderately;

Glandular stomach – reddened, slightly

 

103

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately; Mucous membrane 

                  peeled off;

Glandular stomach – reddened, moderately

 

104

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals - enlarged slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

105

External:

Perineum soiled

 

Internal:

 

Lungs reddened all lobes slightly; Autolytic changes were set in

 

106

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach – reddened, moderately

 

5/

120 mg/kg

 

107

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration markedly; Mucous membrane peeled off;

Glandular stomach – reddened, moderately

 

108

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach – reddened, moderately

 

109

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach - reddened moderately;

Autolytic changes were set in

 

110

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus – small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

5/

120 mg/kg

 

111

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach –reddened, moderately;

Autolytic changes were set in

 

112

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; reddish fluid content present; Ulceration

                  moderately;

Glandular stomach – reddened, slightly

 

113

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; Ulceration, moderately;

Glandular stomach – reddened, slightly

 

114

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals - enlarged in size, slightly;

Stomach - non glandular mucosa thickened slightly; raised whitish foci moderately; Ulceration slightly; Glandular stomach - reddened slightly

 

115

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Stomach – distended, slightly; Fore stomach raised whitish foci

                  markedly; Ulceration moderately;

Glandular stomach - reddened slightly

 

5/

120 mg/kg

 

116

External:

Perineum soiled; Rigor mortis present

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen - enlarged in size, slightly;

Stomach - distended moderately; Fore stomach raised whitish foci

                 markedly; Ulceration, markedly;

Glandular stomach - reddened moderately

 

117

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals - enlarged in size, slightly;

Stomach - non glandular mucosa thickened slightly; raised whitish foci moderately; Ulceration, moderately; Glandular stomach - reddened slightly

 

118

External:

Perineum soiled

 

Internal:

 

Thymus - small in size, slightly;

Adrenals – enlarged, slightly;

Spleen – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  markedly; Ulceration, markedly;

Glandular stomach - reddened moderately;

Autolytic changes were set in

 

119

External:

Perineum soiled; Rigor mortis present; Nostrils reddish discharge slightly

 

Internal:

 

Thymus - small in size, moderately;

Adrenals – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular & non glandular – reddened, moderately

 

120

External:

Perineum soiled; Rigor mortis present; Nostrils reddish discharge slightly

 

Internal:

 

Thymus - small in size, moderately;

Adrenals – enlarged, slightly;

Stomach – distended, moderately; Fore stomach raised whitish foci

                  moderately; Ulceration, moderately;

Glandular & non glandular – reddened, moderately

 

Conclusions:
Pregnant females (24/dose group) were dosed by oral gavage at 0, 20, 40, 80 and 120 mg/kg bw /d solved in water as vehicle beginning at day 6 of pregnancy up to day 19 of pregnancy. On day 20 all females were sacrificed by caesarian section. Fetuses were counted, weighted, sex determined and divided in two groups by randomization. One group of fetuses was selected for visceral examination the second group was prepared for skeletal examination.

Dullness, diarrhoea, piloerection and epistaxis (two females) were observed in 24 out of 24 female animals treated at 120 mg/kg body weight (high dose). All animals of the high dose group died during day 8 to day 16 of gestation (i.e. between day 4 and day 12 of treatment). Gross pathological local findings of the decedent dams at stomach, fore-stomach and glandular stomach are considered a direct consequence of the chemical property of the test substance, namely being a cationic surfactant. These findings are:
- Stomach – distended, moderately to markedly;
- Fore stomach raised whitish foci, moderately to markedly;
- Ulceration moderately to markedly;
- Mucous membrane peeled off in some females;
- Glandular stomach – reddened, slightly to moderately.
No mortality or clinical signs of toxicity or macroscopic changes were observed in all animals dosed up to 80 mg/kg bw/d.

The evaluation of the reproductive organs of all dams dosed up to 80 mg/kg bw/d, pre- and post-implantation loss, gravid uterine weight, litter size, viability of the fetuses, foetus weight per litter and foetus weight, sex ratio of the fetuses and the external, visceral and skeletal examination of the fetuses revealed that HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the dose of 80 mg/kg body weight. Hence, under the present experimental conditions, HYEQS is not a teratogen.
Executive summary:

Pregnant females (24/dose group) were dosed by oral gavage at 0, 20, 40, 80 and 120 mg/kg bw /d solved in water as vehicle beginning at day 6 of pregnancy up to day 19 of pregnancy. On day 20 all females were sacrificed by caesarian section. Fetuses were counted, weighted, sex determined and divided in two groups by randomization. One group of fetuses was selected for visceral examination the second group was prepared for skeletal examination.

Dullness, diarrhoea, piloerection and epistaxis (two females) were observed in 24 out of 24 female animals treated at 120 mg/kg body weight (high dose). All animals of the high dose group died during day 8 to day 16 of gestation (i.e. between day 4 and day 12 of treatment). Gross pathological local findings at stomach, forestomach and glandular stomach are considered a direct consequence of the chemical property of the test substance. No mortality or clinical signs of toxicity were observed in all animals dosed up to 80 mg/kg bw/d.

The evaluation of the reproductive organs of the dams, pre- and post-implantation loss, gravid uterine weight, litter size, viability of the foetuses, foetus weight per litter and foetus weight, sex ratio of the foetuses and the external, visceral and skeletal examination of the foetuses revealed that HYEQS did not produce any malformations and no maternal or embryo-fetal toxicity up to the dose of 80 mg/kg bw/d. Hence, under the present experimental conditions HYEQS is not a teratogen.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Scientific well performed GLP compliant OECD 414 study.
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

The assessment of the endpoint developmental toxicity/teratogenicity is based on an available OECD 422 study with HYEQS as well as on an OECD 414 (rat) with HYEQS.

In addition there is a supportive OECD 414 (rat) study with the analogue substance C8-C10 alkyl dimethyl hydroxyethyl ammonium chloride available. The NOAEL (maternal toxicity) in this study was determined 50 mg/kg bw/day (OECD414 study) based on increased incidences of adverse clinical observations, reductions in body weight gain and feed consumption values and mortalities at the highest dose level of 100 mg/kg bw/day. The NOAEL with regard to developmental toxicity was also determined 50 mg/kg bw/day (OECD 414 study) based on a reduction in fetal body weights and delays in skeletal ossifications in the highest dose group. The NOAEL maternal and developmental toxicity of 50 mg/kg bw/day support the results of the registration substance HYEQS.

In the OECD 422 study performed with the registration substance HYEQS, findings at necropsy of the 60 mg/kg bw/d dose group after about 53 days of daily oral application are based on local effects due to the corrosive properties of the HYEQS. The NO(A)EL for toxicity to reproduction in this study was placed at 60 mg/kg bw/day.

For assessment purposes the no observed adverse effect level NOAEL for developmental toxicity of 80 mg/kg bw/day from the OECD 414 guideline study (rat) with the registration substance HYEQS as most relevant test is taken for the CSA.

Justification for classification or non-classification

Based on data from a prenatal developmental study in Wistar rats OECD 414 performed in 2016 with HYEQS, no classification indicated.