Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl (3,6,9,12-tetraoxahexadecyl)adipate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May - 22 Sept 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May - 22 Sept 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Government of India, Department of Science and Technology, National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

recommended by regulatory agencies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Biosciences, Inc., USA, through its representative Vivo Bio Tech Ltd., Telangana, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation:males: 303 - 389 g (mean 356.30 g) / females: 210 - 242 g (mean: 226.33 g)
- Housing: Maximum three animals per sex and cage in solid bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob.
- Diet (ad libitum): 'Altromin' brand extruded pelleted rat feed manufactured by Altromin Spezialfutter GmbH & Co. KG, Germany
- Water (ad libitum): Potable water, passed through 'Aquaguard' water filter, and subjected to ultra violet irradiation
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: daily
Dosing formulations were prepared freshly each day before dosing. Corn oil was used as vehicle control item. The test item was completely miscible in corn oil. Required amount of test item was weighed on an analytical balance, then corn oil was added gradually to achieve appropriate concentrations i.e. 20, 60 and 200 mg/mL to meet dosage level requirements.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: 040516
- Date of Manufacture: 4 May 2016
- Date of Expiry: 3 May 2018
- Storage Conditions: room temperature (27 +/- 9 °C)
- Manufacturer: Medcraft International Pvt. Ltd. New Delhi, India

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in the vehicle for the highest and lowest concentrations were determined at the Analytical Chemistry Section of the test facility in a separate study. A validated HPLC-UV method was used for identification and quantification of the test item in the vehicle. The HPLC method has been developed in the test facility. Test item formulations were subjected to verification for highest and lowest concentrations twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study.

Duration of treatment / exposure:

Males: minimum four weeks (minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating).
Females: throughout the study (two weeks prior to mating, variable time to conception, during pregnancy and thirteen days after delivery).

Frequency of treatment:

7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Males: 10
Females: 15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a 14-day dose range finder study. Groups of five male and five female Wistar rats were administered the test item by oral gavage daily at the doses of 125, 500 and 1000 mg/kg bw for 14 days and were sacrificed on day 15 to evaluate its toxicity. A concurrent vehicle control group receiving corn oil at the dose of 5 mL/kg was also maintained. The test item did not induce any mortality and treatment-related clinical abnormalities in rats treated at and up to the dose of 1000 mg/kg bw/day. No mortality or abnormal clinical signs were observed in vehicle control group animals. Hence, dose levels of 100, 300, and 1000 mg/kg bw/day were selected for the main study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons), and once a week thereafter
- Detailed clinical observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included, changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behavior were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition and at day 4 and day 13 post-partum.

FOOD CONSUMPTION: Yes
- During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation day 0, 7, 14 and 20 and in lactation on days 4 and 13. Food consumption during mating period was not measured.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken before start of treatment and after completion of pre-mating period
- Anaesthetic used for blood collection: Yes, light CO2 anaesthesia
- Animals fasted: Yes, overnight but with access to water ad libitum
- How many animals: 5 males and 5 females randomly selected from each group before start of treatment and 5 males and 5 females randomly selected from each group after completion of pre-mating period
- Parameters checked: Haemoglobin (Hb), Haematocrit (PCV), Erythrocyte Count, Total (Total RBC), Reticulocytes, Total Leukocyte Count (Total WBC), Platelet Count, Total (Platelet), Prothrombin time as a measure of blood clotting (seconds), Differential Leukocyte (WBC) Count (Leucocytes were differentiated as Neutrophils (N), Lymphocytes (L), Eosinophils (E), Monocytes (M) and Basophils (B)), General blood picture (Blood smear was assessed for abnormal and immature cells during microscopy).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after termination
- Animals fasted: Not specified
- How many animals: 5 males and 5 females randomly selected from each group
- Parameters checked: Sodium, Potassium, Glucose, Total Cholesterol, Urea, Creatinine, Total Protein, Albumin, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, Thyroid Hormone (total), Urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at one time during the study following the daily dose administration (males shortly before the scheduled sacrifice and females during the last week of lactation shortly before scheduled sacrifice)
- Dose groups that were examined: five P generation males and five P generation females randomly selected from each group
- Battery of functions tested (Examinations in home-cage and open field): Posture / Movement, Respiration, Palpebral closure, Lacrimation, Salivation, Skin and hair coat, Urination, Defecation, Locomotor activity, Rearing, Gait
- Battery of functions tested (Manipulative examination / Responses to stimuli): Tactile (touch) response, Response to non receptive stimuli (tail pinch), Pupil response to light, Proprioception - Righting reflex, Auditory response, Head shaking, Landing foot splay, Grip strength (fore limb and hind limb)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Tissues and organs examined: Gross lesions, Brain (cerebrum, cerebellum, midbrain), Spinal cord, Eyes, Thyroid, Parathyroid, Spleen, Thymus, Adrenals, Trachea, Lungs, Heart, Oesophagus, Stomach, Duodenum, Jejunum, Terminal ileum (with peyer's patch), Colon, Rectum, Liver, Kidneys, Urinary bladder, Prostate, Seminal vesicle with coagulating glands, Epididymides, Testes, Ovaries, Uterus with cervix, Vagina, Skeletal muscles, Sciatic nerve, Bone marrow (smear), Mesenteric lymph node, Axillary lymph node, Bone-femur, Levator ani plus bulbocavernosus muscle complex, Glans penis, Cowpers gland
- Further details: All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded. All the tissues listed from five adult males and females, randomly selected from each group, were preserved in 10% neutral buffered formalin. Testes were collected in Modified Davidson's fluid. The tunica albuginea of the testes was gently punctured at both poles of the organ with a needle to permit rapid penetration of the fixative. Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle. Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

HISTOPATHOLOGY: Yes
- Tissues and organs examined: Gross lesions, Brain (cerebrum, cerebellum, midbrain), Spinal cord, Eyes, Thyroid, Parathyroid, Spleen, Thymus, Adrenals, Trachea, Lungs, Heart, Oesophagus, Stomach, Duodenum, Jejunum, Terminal ileum (with peyer's patch), Colon, Rectum, Liver, Kidneys, Urinary bladder, Prostate, Seminal vesicle with coagulating glands, Epididymides, Testes, Ovaries, Uterus with cervix, Vagina, Skeletal muscles, Sciatic nerve, Bone marrow (smear), Mesenteric lymph node, Axillary lymph node, Bone-femur, Levator ani plus bulbocavernosus muscle complex, Glans penis, Cowpers gland
- Further details: From five adult males and females, randomly selected of groups G1 (control) and G4 (high dose), sacrificed at termination, the tissues were fixed in 10% neutral buffered formalin, were embedded in paraffin wax, sectioned at 5 µm thickness and stained with haematoxylin and eosin, for microscopic examination. These examinations were not extended to animals of other dosage groups, as treatment-related changes were not observed in the high dose group. In absence of any microscopic alteration in the thyroid glands of adult male and female rats and in absence of any alterations in the values of total T4, microscopic evaluation of thyroid glands from the pups was not carried out.

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23). For statistical analysis, the litter was used as the basic sampling unit. The following statistical methods were used to analyse the various parameters:

- One-way ANOVA followed by Dunnett's test
- Kruskal -Wallis followed by Mann- Whitney test
- Chi-Square/fisher test

The results of these statistical analyses were assessed at 5% level of significance (p = 0.05) and designated as significantly higher (S+) / lower (S-) than control values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly low (p < 0.05) mean percentage PCV in high dose males and significantly high (p < 0.05) mean reticulocyte count percentage in high dose females were considered incidental because there was no dose-dependant change and the values were within respective historical control range.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A few instances of statistically significant (p < 0.05) changes compared to that of control organ weights were noticed in males and were considered incidental as they were within historical control ranges. Observations for males include:
- Mean absolute weight of adrenals 0.044 ± 0.001 g; Historical data range 0.030 to 0.072 g
- Mean absolute weight of spleen 0.73 ± 0.07 g; Historical data range 0.43 to 1.09 g
- Mean relative weight of spleen 0.20 ± 0.02 g; Historical data range 0.16 to 0.29 g
Moreover, a few instances of statistically significant (p < 0.05) changes were noticed in female rats and were considered incidental. They were, increase in absolute and relative weight of adrenals in (G2) low dose group and increase in only relative weight of adrenals in (G3) mid dose group rats. The effect was not evident in the high dose group rats and hence, these changes were not considered to be treatment-related as there were no dose-related responses. Also the relative and absolute weights of adrenals were well within the historical control range of female rats of similar age group from the test facility, though not from reproduction study.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Some incidental and spontaneous lesions observed in animals from the high dose group (1000 mg/kg bw/day) comprised of bile duct hyperplasia in liver; ectopic thymus in thyroid and lymphocytic cell infiltration in prostate. All these changes were of minimal severity and were solitary in nature. These changes are commonly observed in rats and as they are known to be spontaneous in nature were not considered to be treatment-related. Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment-related morphological alterations.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS:
Treatment did not induce any adverse clinical signs at any dose levels. No abortion/premature deliveries were observed in any of the dose levels during the study period.

MORTALITIY:
No mortality occurred during the study period that was considered to be related to treatment with the test substance. All animals survived throughout the treatment period of the study.

FUNCTIONAL OBSERVATIONS (NEUROLOGICAL EXAMINATION):
The functional observations did not reveal any remarkable and treatment-related incidence of neurological abnormalities. In addition, no findings indicative of a neurotoxic potential of the test item were observed.

BODY WEIGHT AND WEIGHT GAIN:
The test item at and up to the highest dose of 1000 mg/kg bw/day did not have any remarkable and adverse effects on the weight in male and female rats.

FOOD CONSUMPTION:
Treatment of male and female rats at and up to the highest dose of 1000 mg/kg bw/day did not induce any remarkable and adverse effects on the daily food intake of animals. The values of mean daily food intake by rats treated with test item did not differ significantly (p > 0.05) from those of the concurrent control group females during the pre-mating period, the gestation period and the post-natal lactation period.

HAEMATOLOGY:
No toxicologically relevant changes occurred in haematological parameters of treated rats. Few changes like significantly lower (p < 0.05) mean percentage PCV in high dose males and significantly higher (p < 0.05) mean reticulocyte count percentage in high dose females were considered incidental as there was no dose-dependent change and the values were within respective historical control range: Mean PCV of high dose males was 42.32 ± 2.19%, which is well within historical control range (41.11 to 48%) and the mean reticulocyte count percentage in high dose females was 4.42 ± 1.36% which is well within historical control range (1.83 to 4.58%).

CLINICAL CHEMISTRY:
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a treatment-related trend. At the end of treatment these changes consisted of lower potassium and alkaline phosphatase in males at 300 mg/kg bw/day, and higher urea levels in females at 300 mg/kg bw/day.

ORGAN WEIGHTS:
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. A few instances of statistically significant (p < 0.05) changes compared to that of control organ weights were noticed in males and were considered incidental as they were within historical control ranges. Differences in organ weights were observed in mean absolute weights of adrenals and spleen and the mean relative weight of spleen. Moreover, a few instances of statistically significant (p < 0.05) changes were noticed in female rats and were considered incidental. They were, increase in absolute and relative weight of adrenals in (G2) low dose group and increase in only relative weight of adrenals in (G3) mid dose group rats. The effect was not evident in the high dose group rats and hence, these changes were not considered to be treatment-related as there were no dose-related responses. Also the relative and absolute weights of adrenals were well within the historical control range of female rats of similar age group from the test facility, though not from reproduction study.

GROSS PATHOLOGY:
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations in tissues of treated rats. Moreover, examination of the reproductive organs of male and female rats did not reveal any treatment-related morphological changes.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Histopathological examinations of adult male and female rats randomly selected from control group and high dose groups did not reveal any significant and treatment-related histopathological alterations. Some incidental and spontaneous lesions observed in animals from the high dose group (1000 mg/kg bw/day) include bile duct hyperplasia in liver; ectopic thymus in thyroid and lymphocytic cell infiltration in prostate. All changes observed were of minimal severity and were solitary in nature. These changes are commonly observed in rats and as they are known to be spontaneous in nature were not considered to be treatment-related. Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment-related morphological alterations. Based on the results of the haematology investigation, especially of Total and Differential Leucocyte Counts and general blood picture along with results of organ weights and histopathology of thymus and spleen, there is no evidence of any immunological effects.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.

Critical effects observed:

no

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Government of India, Department of Science and Technology, National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl(3,6,9,12-tetraoxahexadecyl)adipate

Molecular formula:

C26H50O10 C28H54O11

IUPAC Name:

Reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and 2-[2-(2-butoxyethoxy)ethoxy]ethyl(3,6,9,12-tetraoxahexadecyl)adipate

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

recommended by regulatory agencies

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Biosciences, Inc., USA, through its representative Vivo Bio Tech Ltd., Telangana, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation:males: 303 - 389 g (mean 356.30 g) / females: 210 - 242 g (mean: 226.33 g)
- Housing: Maximum three animals per sex and cage in solid bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob.
- Diet (ad libitum): 'Altromin' brand extruded pelleted rat feed manufactured by Altromin Spezialfutter GmbH & Co. KG, Germany
- Water (ad libitum): Potable water, passed through 'Aquaguard' water filter, and subjected to ultra violet irradiation
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: daily
Dosing formulations were prepared freshly each day before dosing. Corn oil was used as vehicle control item. The test item was completely miscible in corn oil. Required amount of test item was weighed on an analytical balance, then corn oil was added gradually to achieve appropriate concentrations i.e. 20, 60 and 200 mg/mL to meet dosage level requirements.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: 040516
- Date of Manufacture: 4 May 2016
- Date of Expiry: 3 May 2018
- Storage Conditions: room temperature (27 +/- 9 °C)
- Manufacturer: Medcraft International Pvt. Ltd. New Delhi, India

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal smear, referred to as Day 0 of pregnancy

In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered. Females showing no evidence of copulation were sacrificed 25 days after the last day of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in the vehicle for the highest and lowest concentrations were determined at the Analytical Chemistry Section of the test facility in a separate study. A validated HPLC-UV method was used for identification and quantification of the test item in the vehicle. The HPLC method has been developed in the test facility. Test item formulations were subjected to verification for highest and lowest concentrations twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study.

Duration of treatment / exposure:

Males: minimum four weeks (minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating).
Females: throughout the study (two weeks prior to mating, variable time to conception, during pregnancy and thirteen days after delivery).

Dosing was continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum total dosing period of 28 days had been completed. They were then sacrificed. Daily dosing of the parental females were continued throughout pregnancy and up to, and including, day 13 post-partum (the day before sacrifice).

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Males: 10
Females: 15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14-day dose range finding study

Examinations

Parental animals: Observations and examinations:

Mating date, confirmation of pregnancy, and delivery day were recorded. The duration of gestation was recorded and was calculated from day 0 of pregnancy. Dams were observed for signs suggestive of abortion or for premature delivery.

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1

Oestrous cyclicity (parental animals):

A total of 60 females was screened for normal oestrous cycle for 2 weeks prior to the treatment period. All females exhibited an oestrous cycle of 4-5 days. Stages of the oestrous cycle were monitored daily in the females randomly assigned to four groups (15 females/group), from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper's glands and glans penis of all male adult animals were weighed on termination of treatment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, Estimation of Thyroid Hormones (Total T4)

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals which delivered: Day 14 post-partum
- Maternal animals which failed to deliver: 25 days after the last day of the mating period

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed on day 13 post-partum.
- Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Blood samples from the day 13 pups were assessed for serum levels of thyroid hormone (T4).

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

Statistics:

See Section 7.5.1

Reproductive indices:

For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of post-natal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Offspring viability indices:

For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of post-natal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly low (p < 0.05) mean percentage PCV in high dose males and significantly high (p < 0.05) mean reticulocyte count percentage in high dose females were considered incidental because there was no dose-dependent change and the values were within respective historical control range.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Some incidental and spontaneous lesions observed in animals from the high dose group (1000 mg/kg) comprised of bile duct hyperplasia in liver; ectopic thymus in thyroid and lymphocytic cell infiltration in prostate. All these changes were of minimal severity and were solitary in nature. These changes are commonly observed in rats and as they are known to be spontaneous in nature were not considered to be treatment related. Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment-related influences on oestrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Details on results (F1)

Embryotoxic / teratogenic effects: no effects observed

MORTALITY AND CLINICAL SIGNS:
Treatment of dams with the test item did not have any adverse effect on the survival of the offspring during the lactation period. The incidence of litters with still-born pups, pups found dead in cage or cannibalized pups was very small and/or comparable across the treated and the control groups. Treatment did not induce any abnormal clinical signs, indicative of systemic toxicity, in offspring during lactation period.

BODY WEIGHT:
The mean litter body weights and mean litter body weight gain did not differ significantly (p > 0.05) from those of the concurrent control group during the post-natal lactation period except in high dose group pups. However, on day 2 of the post-natal lactation period, there was a significantly reduced (p < 0.05) body weight gain. Since the same group pups had gained weight at all other time points up to 13 days which was comparable to that of the control animals at all time points, this finding is considered to be of no toxicological significance.

ANOGENITAL DISTANCE:
No adverse effect on the anogenital distance for male and female pups was observed as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the post-natal day (PND) 0 to PND 4.

NIPPLE RETENTION:
The test item did not influence the nipple retention in male pups at post-natal day 12.

CLINICAL CHEMISTRY - Estimation of Thyroid Hormones (Total T4):
The test item did not induce any changes in the total T4 on day 4 after birth and also on day 13 of lactation.

ORGAN WEIGHTS:
Average absolute weights of thyroid glands from day 13 pups of all treatment group were found to be comparable with those of the control group.

EXTERNAL EXAMINATIONS:
Dead pups and pups killed on day 13 post-partum were carefully examined for external gross abnormalities. The total number of foetuses examined for external examination was 101 in the control group, 102, 100 and 120 in the 100, 300 and 1000 mg/kg bw/day dose groups, respectively. The incidence of normal foetuses and litters observed in this study was 100% in control group and in all treatment groups. There were no any external abnormalities observed in external genitalia or any other organs of pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically relevant effects were observed up to and including the highest dose level

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Summary tables are provided in a pdf document attached to this IUCLID dossier.