Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-decanoic, n-heptanoic, and n-octanoic acids

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May - 05 Sep 2022

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 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 May - 05 Sep 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certifying authority: National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA), Department of Sicence and Technology, Government of India

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The regulatory guidelines for this test has preferred rat among the species of rodents. The Wistar strain has been used for non-clinical safety studies, recommended by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc. (USA) through their technology licensee 'Hylasco Bio-Technology (India) Pvt. Ltd., Telangana,' India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 462 - 510 g, mean 488.8 g (males); 237 - 311 g, mean 275.03 g (females)
- Fasting period before study: no
- Housing: Maximum three per sex / cage in solid [Sized: 42 cm (L) x 29 cm (W) x 19 cm (H)] bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob. Cages were suspended on movable stainless steel racks arranged in such a way that every cage would receive almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week throughout the treatment and observation period. Animals were housed in groups or singly, as follows: Pre-mating period: males and females were housed in groups of maximum two or three per cage per sex. During mating (co-habitation): one male: one female, per cage. Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dams were housed individually.
- Diet: 'Altromin' brand pelleted rat feed (M/s Altromin Spezialfutter GmbH & Co. KG, Germany); ad libitum
- Water: Potable water, passed through ‘Aquaguard’ water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized glass bottles.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The diet has been tested and certified to be free from undesired levels of environmental contaminants. Water is analysed quarterly for potability and once in a year for various environmental contaminants.

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

IN-LIFE DATES: From 17 May To 14 Aug 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared freshly each day before dosing. Corn oil was used as control item. Formulations for different doses varied in concentrations to allow a constant dosage volume of 5 mL/kg bw. The required amount of test item was weighed on analytical balance, then corn oil was added gradually to achieve appropriate concentrations i.e. 50, 100 and 200 mg/mL to meet dosage level requirements.

VEHICLE
- Justification for use and choice of vehicle: The test item's solubility/suspensibility in vehicles water, Tween 80 (aqueous) and aqueous Carboxy methyl cellulose (CMC) was poor / immiscible. Test item was completely miscible in corn oil, therefore corn oil was chosen as a vehicle for dosing formulations. The stability and homogeneity of the test substance in corn oil was demonstrated by analytical verification.
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A method validation study (R/RA4055/AMV-SHA/22) was conducted to determine stability and homogeneity of the test substance in corn oil. The applied method was gas chromatography.
Stability: Formulations at different doses were prepared in corn oil and analysed for determination of the test substance's concentration after specific storage periods. Data on peak area for the test substance's concentrations after specified storage periods were compared with the data obtained before storage.
Results: The % difference in the concentration of test item prepared in corn oil after specified storage period and conditions was found within the acceptance criteria of ± 20% over their initial concentrations. For details, please refer to Table 1 under "Any other information on materials and methods, incl. tables."
Homogeneity: Formulations at different doses were prepared in corn oil and analysed for
determination of the test substance's concentration in the samples drawn from different locations of the container. Data on the test substance's concentrations in samples of test item formulation drawn from top and bottom layer were compared with the data of middle layer. The concentrations of formulation of test item prepared in corn oil at top and bottom layers were found within ± 20% over middle position concentration. For details, please refer to Table 2 under "Any other information on materials and methods, incl. tables".

Duration of treatment / exposure:

males: premating (2 weeks), during the mating period, post mating (until a total dosing period of 28 days)
females: total: 50 - 60 days (2 weeks pre-mating, throughout conception and pregnancy, until 13 days after delivery, up to and including the day before scheduled sacrifice

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

G2

Dose / conc.:

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

Remarks:

G3

Dose / conc.:

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

Remarks:

G4

No. of animals per sex per dose:

10 males and 15 females for treatment and negative (vehicle) control groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a preliminary 14-days dose-range finding study (R/21410/SOR-14-DRF/22), in which the test substance did not induce any adverse effects up to and including the dose of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Dose range finding studies: A 14-days dose-range finding study was conducted (No. P/21410/SOR-14-DRF/22) in Wistar rats, in which 5 animals/sex/dose received 125, 250, 500 or 1000 mg/kg bw/day test substance in corn oil via oral gavage. Mortality/moribundity, clinical signs, body weight, food consumption, gross macroscopic examination and organ weights of kidneys, liver, adrenals, testes, spleen, brain, ovaries and heart were recorded. There were no adverse effects noted in any of the investigated parameters up to and including the highest dose level (1000 mg/kg bw/day) tested.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for signs of toxicity, morbidity and mortality
All signs of illness, together with any behavioural changes or reaction to treatment were observed. Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the study, and weekly thereafter during the treatment period and at termination
These 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: First day of dosing, weekly thereafter and at termination. During pregnancy, females were weighed on gestational days 0, 7, 14, 20 and then within 24 h of parturition (day 1 post-partum), at day 4 and day 13 post-partum.

FOOD CONSUMPTION
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 during lactation on days 4 and 13.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled sacrifice
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes, overnight
- How many animals: 5 randomly selected males and females per dose group (for baseline evaluations prior to treatment start and at the end of the premating period)
- Parameters checked in Table 3 under "Any other information on materials and methods, incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes, overnight
- How many animals: 5 randomly selected males and females per dose group
- Parameters checked in Table 4 under "Any other information on materials and methods, incl. tables" were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: during litter standardization, under sodium thiopentone anesthesia
- Animals fasted: dams only
- How many animals: From 2 of the surplus pups, if possible. Alternatively, from 1 surplus pup; and from 5 randomly selected males and females per dose group (adults, baseline sampling), from all adult males at termination (day 29), and from all dams at termination on lactation day 14. The hormone analyses was carried out on 'Multiskan Go Microplate and Cuvette Spectrophotometer (ELISA Reader-Make- Thermo Scientific)' with commercially available ELISA kits manufactured by Elabscience, WuHan, P.R.C.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: towards the end of dosing period (shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry); females: during lactation (lactation day 6 - 13).
- Dose groups that were examined: 5/sex/dose
- Battery of functions tested: sensory activity, grip strength and motor activity
This neurological examination included:
1. Examinations in home-cage and open field: a. Posture / Movement, b. Respiration, c. Palpebral closure, d. Lacrimation, e. Salivation, f. Skin and hair coat, g. Urination, h. Defecation, i. Locomotor activity, j. Rearing, k. Gait
2. Manipulative examination / Responses to stimuli: a. Tactile (touch) response, b. Response to non receptive stimuli (tail pinch), c. Pupil response to light, d. Proprioception – Righting reflex, e. Auditory response, f. Head shaking, g. Landing foot splay, h. Grip strength (fore limb and hind limb) - (by ‘Grip Strength Meter, Cat. No. 47200 by UGO Basile Biological Research Apparatus’)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Females showing no evidence of copulation were sacrificed 25 days after the last day of the mating period. Dams found sperm positive and failed to deliver by gestation day 24 were weighed and sacrificed by CO2 asphyxiation on their gestation day 25. The dams were subjected to a necropsy examination to observe any gross pathological changes, and the findings were recorded.
Males were sacrificed after the minimum total dosing period of 28 days had been completed.
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.

HISTOPATHOLOGY: Yes
All the tissues listed below, from all adult males and females 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. Histopathology was performed on all listed organs and tissues of selected five males and five females of groups G1 (control) and G4 (high dose), sacrificed at termination. All 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.

Preserved and evaluated tissues: 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, and Cowper's gland.

ORGAN WEIGHTS
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 adult male animals were weighed on termination of treatment (on day 29). Uterus with cervix and ovaries from all adult females were weighed at termination on lactation day 14. From all adult males and females, thyroid glands were preserved and weighed post fixation. At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation.
Trimming was done very carefully and only after fixation to avoid tissue damage. In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.

Optional endpoint(s):

No

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23). For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters. The results of these statistical analyses were assessed at 5% level of significance (p = 0.05) and designated as significantly higher / lower than control values, using *.
For details, please see Table 5 under "Any other information on materials and methods, incl. tables."

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse clinical signs at any dose level.

Mortality:

no mortality observed

Description (incidence):

There was no incidence of mortality amongst the rats treated with the test substance at any of the dose levels in both males and females. No incidences of moribund or found dead dams were observed during the study period. All animals survived throughout the period of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant reduction in body weight and body weight gain of male rats as compared to the control group was observed at 1000 mg/kg bw/day after two weeks following the treatment until termination. Although, reduction in body weight gain at fourth week was not statistically significant when compared to the control group. Further, this change in body weight was visible by statistical significant reduction in overall body weight gain over four weeks as compared to those of control group.
For details on male body weight and body weight gain, please refer to Tables 1 and 2, respectively, under "Any other information on results incl. tables.".

Body weight and body weight gain in female rats was found to be comparable to that of the control rats during premating and mating period at and up to 1000 mg/kg bw/day except statistically significant but transient increase in body weight gain at the end of first week only in mid-dose group (G3 - 500 mg/kg bw/day). The effect was considered as incidental. For details, please refer to Table 3 under "Any other information on results incl. tables." There was no significant difference (p>0.05) in the mean body weights of control and treated groups of females during the period of gestation. The mean body weight of female rats as measured on gestation days 0, 7, 14 and 20 and the mean body weight changes computed between gestation days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats. During all other time periods and in all other dose groups, body weight and body weight gain were comparable to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in food consumption at any dose level, in either sex, or during any of the treatment periods.

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):

A statistically significant increase was observed in MCV in male rats at 1000 mg/kg bw/day. The values are well within historical control data of the laboratory. Further, in absence of any other corroborative alterations this change was considered as incidental.

There were no changes in any other parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistical significant increase was observed in total cholesterol (CHOL) in male rats only of 250 mg/kg bw/day dose level. This change was considered as incidental as there were no dose-dependent changes.

There were no changes in any other parameters, including thyroid hormone (T4) measurements.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- Vaginal smears / estrous cycle
- Genital abnormalities
- T4 levels (pups and parental animals, males/females)
- Anogenital distance
- Nipple development (males)
- Reproductive / viability parameters: gestation length, pre- / post-implantation loss, number of corpora lutea, number of implantations, gestation length, sex ratio, pup / litter weights
- Organ weights and histopathology: adrenals, brain, Cowper's gland, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex, liver, ovaries, prostate, seminal vesicles (including coagulating glands), testes, thyroid gland, uterus (with cervix)
- Histopathology: adrenals, brain, Cowper's gland, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex, liver, ovaries, prostate, seminal vesicles (including coagulating glands), testes, thyroid gland, parathyroid gland, uterus (with cervix), and vagina
For details, please refer to the respective result fields in the present RSS, as well as the RSS cross-link in Chapter 7.8.1

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no remarkable and test item related incidence of neurological abnormalities. Also no findings indicative of a neurotoxic potential of the test item were encountered during these examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase (p<0.05) in relative weight of testes in the high dose gropu (G4 - 1000 mg/kg/bw/day) and of epididymis in mid dose (G3 - 500 mg/kg/bw/day) and high dose (G4 - 1000 mg/kg/bw/day) was observed. Increase in relative weight of epididymis was not dose dependent. Furthermore, due to lack of corroborative effects in gross and histopathology, increase in relative weight of testes of epididymis was considered incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The test item, at and up to the dose level of 1000 mg/kg bw/day, did not induce any remarkable and test item related gross pathological alterations in any of the tissues of treated rats, as evident by the detailed necropsy examination carried out at termination. Gross pathological examination of the reproductive organs of male and female rats did not reveal any test item related morphological alterations.

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 control and high dose group
(1000 mg/kg bw/day) included peribronchial lymphoid hyperplasia in lungs, multifocal and minimal cortical vacuolation in adrenals, dilated glands in stomach, sub-mucosal lymphoid hyperplasia in rectum and minimal presence of hyaline casts in kidneys. All these changes were of minimal severity and mostly solitary. These changes are commonly observed in rats of this age and were not considered to be test item related. Histopathological examination of the reproductive organs of male and female rats did not reveal any test item related morphological alterations.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect observed up to and including the highest dose level tested.

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effect observed at this dose level.

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Table 1: Summary data on body weight in males during the premating and mating period

Group	Dose (mg/kg bw/day)	Study Day	Treatment Period
			0	7	14	21	28
Vehicle control: corn oil
G1 5 mL/kg bw/day		Mean	489.3	515.2	530.7	541.5	550.4
		± S. D.	12.01	18.41	17.46	22.04	26.61
		n	10	10	10	10	10
Test item
G2	250	Mean	488.9	513.8	532.7	542.8	542.5
		± S. D.	13.67	20.46	21.41	16.88	16.93
		n	10	10	10	10	10
G3	500	Mean	488.0	518.2	530.7	533.1	539.4
		± S. D.	8.45	9.50	13.29	13.36	13.72
		n	10	10	10	10	10
G4	1000	Mean	489.00	513.30	515.00	491.50*	479.30*
		± S. D.	17.31	19.71	35.40	49.14	51.62
		n	10	10	10	10	10

* p<0.05 compared to control group

 

Table 2: Summary data on body weight gain in males during premating and mating period

Group	Dose (mg/kg bw/day)		Study Days
			0-7	7-14	14-21	21-28	0-28
Vehicle control: corn oil
G1  5 mL/kg bw/day		Mean	5.28	3.03	2.03	1.64	12.52
		± S. D.	1.99	1.35	2.17	2.30	5.35
		n	10	10	10	10	10
Test item
G2	250	Mean	5.08	3.68	1.94	-0.05	10.98
		± S. D.	2.39	1.34	1.74	1.48	2.59
		n	10	10	10	10	10
G3	500	Mean	6.2	2.41	0.46	1.19	10.54
		± S. D.	1.55	1.39	1.37	1.01	2.29
		n	10	10	10	10	10
G4	1000	Mean	4.97	0.32	-4.27*	-2.19	-1.76*
		± S. D.	1.86	5.47	10.13	9.40	11.98
		n	10	10	10	10	10

*: p<0.05 compared to control group

 

Table 3: Summary data for body weight gain in females during the premating period

Group	Dose (mg/kg bw/day)	Study Days
			0-7	7-14	0-14
Vehicle control: corn oil
G1		Mean	-0.23	2.21	1.97
		± S. D.	1.42	2.10	2.34
		n	15	15	15
Test item
G2	250	Mean	0.97	3.05	4.05
		± S. D.	1.66	1.56	2.05
		n	15	15	15
G3	500	Mean	4.46*	3.50	4.46
		± S. D.	3.97	1.57	3.97
		n	15	15	15
G4	1000	Mean	-0.69	3.18	2.49
		± S. D.	3.16	2.80	4.56
		n	15	15	15

* p<0.05 compared to control group

Conclusions:

In the present combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, which was conducted according to OECD 422 and compliant with GLP, Wistar rats were exposed to 0 (control), 250, 500 and 100 mg/kg bw/day test substance via daily oral gavage, covering pre-mating, mating, gestation, lactation periods, up to day 13 post-partum (females) or day 28 after mating (males).
There were no adverse effects on developmental and reproductive parameters, neither in the parents, nor in the offspring. A decrease in body weight and body weight gain was observed in males during premating and mating period at the top dose of 1000 mg/kg bw/day. No other adverse effects were observed. Therefore, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of the test substance in Wistar rats, for systemic toxicity following oral administration for a period of four weeks for male rats was found to be 500 mg/kg bw/day and for a period of 50 to 60 days for females was found to be 1000 mg/kg bw/day. The No-Observed-Adverse-Effect-Level (NOAEL) of EC 453-490-7 in Wistar rats for reproductive and developmental toxicity was found to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

453-490-7

EC Name:

-

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

2-[(decanoyloxy)methyl]-3-[3-(heptanoyloxy)-2-[(octanoyloxy)methyl]-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propoxy]-2-[(octanoyloxy)methyl]propyl decanoate; 3-[3-(heptanoyloxy)-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy]-2-[(heptanoyloxy)methyl]-2-[(octanoyloxy)methyl]propyl decanoate

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The regulatory guidelines for this test has preferred rat among the species of rodents. The Wistar strain has been used for non-clinical safety studies, recommended by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc. (USA) through their technology licensee 'Hylasco Bio-Technology (India) Pvt. Ltd., Telangana,' India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 462 - 510 g, mean 488.8 g (males); 237 - 311 g, mean 275.03 g (females)
- Fasting period before study: no
- Housing: Maximum three per sex / cage in solid [Sized: 42 cm (L) x 29 cm (W) x 19 cm (H)] bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob. Cages were suspended on movable stainless steel racks arranged in such a way that every cage would receive almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week throughout the treatment and observation period. Animals were housed in groups or singly, as follows: Pre-mating period: males and females were housed in groups of maximum two or three per cage per sex. During mating (co-habitation): one male: one female, per cage. Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dams were housed individually.
- Diet: 'Altromin' brand pelleted rat feed (M/s Altromin Spezialfutter GmbH & Co. KG, Germany); ad libitum
- Water: Potable water, passed through ‘Aquaguard’ water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized glass bottles.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The diet has been tested and certified to be free from undesired levels of environmental contaminants. Water is analysed quarterly for potability and once in a year for various environmental contaminants.

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

IN-LIFE DATES: From 17 May To 14 Aug 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared freshly each day before dosing. Corn oil was used as control item. Formulations for different doses varied in concentrations to allow a constant dosage volume of 5 mL/kg bw. The required amount of test item was weighed on analytical balance, then corn oil was added gradually to achieve appropriate concentrations i.e. 50, 100 and 200 mg/mL to meet dosage level requirements.

VEHICLE
- Justification for use and choice of vehicle: The test item's solubility/suspensibility in vehicles water, Tween 80 (aqueous) and aqueous Carboxy methyl cellulose (CMC) was poor / immiscible. Test item was completely miscible in corn oil, therefore corn oil was chosen as a vehicle for dosing formulations. The stability and homogeneity of the test substance in corn oil was demonstrated by analytical verification.
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 1 week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A method validation study (R/RA4055/AMV-SHA/22) was conducted to determine stability and homogeneity of the test substance in corn oil. The applied method was gas chromatography.
Stability: Formulations at different doses were prepared in corn oil and analysed for determination of the test substance's concentration after specific storage periods. Data on peak area for the test substance's concentrations after specified storage periods were compared with the data obtained before storage.
Results: The % difference in the concentration of test item prepared in corn oil after specified storage period and conditions was found within the acceptance criteria of ± 20% over their initial concentrations. For details, please refer to Table 1 under "Any other information on materials and methods, incl. tables."
Homogeneity: Formulations at different doses were prepared in corn oil and analysed for
determination of the test substance's concentration in the samples drawn from different locations of the container. Data on the test substance's concentrations in samples of test item formulation drawn from top and bottom layer were compared with the data of middle layer. The concentrations of formulation of test item prepared in corn oil at top and bottom layers were found within ± 20% over middle position concentration. For details, please refer to Table 2 under "Any other information on materials and methods, incl. tables".

Duration of treatment / exposure:

males: premating (2 weeks), during the mating period, post mating (until a total dosing period of 28 days)
females: total: 50 - 60 days (2 weeks pre-mating, throughout conception and pregnancy, until 13 days after delivery, up to and including the day before scheduled sacrifice

Frequency of treatment:

once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 13 - 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day

Remarks:

G2

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

G3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G4

No. of animals per sex per dose:

10 males and 15 females for treatment and negative (vehicle) control groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a preliminary 14-days dose-range finding study (R/21410/SOR-14-DRF/22), in which the test substance did not induce any adverse effects up to and including the dose of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Dose range finding studies: A 14-days dose-range finding study was conducted (No. P/21410/SOR-14-DRF/22) in Wistar rats, in which 5 animals/sex/dose received 125, 250, 500 or 1000 mg/kg bw/day test substance in corn oil via oral gavage. Mortality/moribundity, clinical signs, body weight, food consumption, gross macroscopic examination and organ weights of kidneys, liver, adrenals, testes, spleen, brain, ovaries and heart were recorded. There were no adverse effects noted in any of the investigated parameters up to and including the highest dose level (1000 mg/kg bw/day) tested.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for signs of toxicity, morbidity and mortality
All signs of illness, together with any behavioural changes or reaction to treatment were observed. Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the study, and weekly thereafter during the treatment period and at termination
These 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: First day of dosing, weekly thereafter and at termination. During pregnancy, females were weighed on gestational days 0, 7, 14, 20 and then within 24 h of parturition (day 1 post-partum), at day 4 and day 13 post-partum.

FOOD CONSUMPTION
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 during lactation on days 4 and 13.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled sacrifice
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes, overnight
- How many animals: 5 randomly selected males and females per dose group (for baseline evaluations prior to treatment start and at the end of the premating period)
- Parameters checked in Table 3 under "Any other information on materials and methods, incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes, overnight
- How many animals: 5 randomly selected males and females per dose group
- Parameters checked in Table 4 under "Any other information on materials and methods, incl. tables" were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: during litter standardization, under sodium thiopentone anesthesia
- Animals fasted: dams only
- How many animals: From 2 of the surplus pups, if possible. Alternatively, from 1 surplus pup; and from 5 randomly selected males and females per dose group (adults, baseline sampling), from all adult males at termination (day 29), and from all dams at termination on lactation day 14. The hormone analyses was carried out on 'Multiskan Go Microplate and Cuvette Spectrophotometer (ELISA Reader-Make- Thermo Scientific)' with commercially available ELISA kits manufactured by Elabscience, WuHan, P.R.C.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: towards the end of dosing period (shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry); females: during lactation (lactation day 6 - 13).
- Dose groups that were examined: 5/sex/dose
- Battery of functions tested: sensory activity, grip strength and motor activity
This neurological examination included:
1. Examinations in home-cage and open field: a. Posture / Movement, b. Respiration, c. Palpebral closure, d. Lacrimation, e. Salivation, f. Skin and hair coat, g. Urination, h. Defecation, i. Locomotor activity, j. Rearing, k. Gait
2. Manipulative examination / Responses to stimuli: a. Tactile (touch) response, b. Response to non receptive stimuli (tail pinch), c. Pupil response to light, d. Proprioception – Righting reflex, e. Auditory response, f. Head shaking, g. Landing foot splay, h. Grip strength (fore limb and hind limb) - (by ‘Grip Strength Meter, Cat. No. 47200 by UGO Basile Biological Research Apparatus’)

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Vaginal smears of all females were monitored daily during pretreatment, premating treatment period and during mating period until evidence of mating. Females that failed to exhibit typical 4 or 5-day cycles were not included in the study. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Not examined.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups, pooled, and used for determination of serum T4 levels. Whenever the numbers of male or female pups were less than four of each sex per litter, partial adjustment (for example, five males and three females) was followed. No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there was only one pup available above the normal litter size, only one pup was eliminated and used for blood collection for serum T4 assessments. When litter size was not adjusted, two pups per litter were sacrificed on day 4 after birth and blood samples were taken for measurement of serum thyroid hormone concentrations. When possible the two pups per litter sacrificed were female pups to reserve male pups for nipple retention evaluations, except in the event that removing these pups leaves no remaining females for assessment at termination.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), presence of gross anomalies, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which was examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
Dead pups were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at the end of the treatment period (28 days of dosing).
- Maternal animals: Females showing no-evidence of copulation / mating were sacrificed 25 days after last day of the mating period. Dams found sperm-positive and failed to deliver by gestation day 24 were weighed and sacrificed by CO2 asphyxiation on their gestation day 25. The dams were subjected to a necropsy examination to observe any gross pathological changes, and the findings were recorded. All surviving animals were killed at the end of the treatment period (day 13 post-partum).

GROSS NECROPSY
- 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.

HISTOPATHOLOGY
All the tissues listed below, from all adult males and females 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. Histopathology was performed on all listed organs and tissues of selected five males and five females of groups G1 (control) and G4 (high dose), sacrificed at termination. All 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.

Preserved and evaluated tissues: 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, and Cowpers gland.


ORGAN WEIGHTS
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 adult male animals were weighed on termination of treatment (on day 29). Uterus with cervix and ovaries from all adult females were weighed at termination on lactation day 14. From all adult males and females, thyroid glands were preserved and weighed post fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.

Postmortem examinations (offspring):

SACRIFICE
Pups were killed on day 13 post-partum.

GROSS NECROPSY
Pups sacrificed on day 13 post-partum (and dead pups) 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 / ORGAN WEIGTHS
At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation. 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. Thyroids were, however, weighed.

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23). For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters. The results of these statistical analyses were assessed at 5% level of significance (p = 0.05) and designated as significantly higher / lower than control values, using *.
For details, please see Table 5 under "Any other information on materials and methods, incl. tables."

Reproductive indices:

Not specified.

Offspring viability indices:

live birth index = (number of live foetus on day 0 / number of fetuses born X 100)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse clinical signs at any dose levels.

Mortality:

no mortality observed

Description (incidence):

There was no incidence of mortality amongst the rats treated with the test substance at any of the dose levels in both males and females. No incidences of moribund or found dead dams were observed during the study period. All animals survived throughout the period of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant reduction in body weight and body weight gain of male rats as compared to the control group was observed at 1000 mg/kg bw/day after two weeks following the treatment until termination. Although, reduction in body weight gain at fourth week was not statistically significant when compared to the control group. Further, this change in body weight was visible by statistical significant reduction in overall body weight gain over four weeks as compared to those of control group.
For details on male body weight and body weight gain, please refer to Tables 1 and 2, respectively, under "Any other information on results incl. tables.".

Body weight and body weight gain in female rats was found to be comparable to that of the control rats during premating and mating period at and up to 1000 mg/kg bw/day except statistically significant but transient increase in body weight gain at the end of first week only in mid-dose group (G3 - 500 mg/kg bw/day). The effect was considered as incidental. For details, please refer to Table 3 under "Any other information on results incl. tables." There was no significant difference (p>0.05) in the mean body weights of control and treated groups of females during the period of gestation. The mean body weight of female rats as measured on gestation days 0, 7, 14 and 20 and the mean body weight changes computed between gestation days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats. During all other time periods and in all other dose groups, body weight and body weight gain were comparable to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in food consumption at any dose level, in either sex, or during any of the treatment periods.

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):

A statistically significant increase was observed in MCV in male rats at 1000 mg/kg bw/day. The values are well within historical control data of the laboratory. Further, in absence of any other corroborative alterations this change was considered as incidental.

There were no changes in any other parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistical significant increase was observed in total cholesterol (CHOL) in male rats only of 250 mg/kg bw/day dose level. This change was considered as incidental as there were no dose-dependent changes.

There were no changes in any other parameters, including thyroid hormone (T4) measurements.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study:
- Vaginal smears / estrous cycle
- Genital abnormalities
- T4 levels (pups and parental animals, males/females)
- Anogenital distance
- Nipple development (males)
- Reproductive / viability parameters: gestation length, pre- / post-implantation loss, number of corpora lutea, number of implantations, gestation length, sex ratio, pup / litter weights
- Organ weights and histopathology: adrenals, brain, Cowper's gland, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex, liver, ovaries, prostate, seminal vesicles (including coagulating glands), testes, thyroid gland, uterus (with cervix)
- Histopathology: adrenals, brain, Cowper's gland, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex, liver, ovaries, prostate, seminal vesicles (including coagulating glands), testes, thyroid gland, parathyroid gland, uterus (with cervix), and vagina
For details, please refer to the respective result fields in the present RSS.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no remarkable and test item related incidence of neurological abnormalities. Also no findings indicative of a neurotoxic potential of the test item were encountered during these examinations.

Immunological findings:

not examined

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 control and high dose group
(1000 mg/kg bw/day) included peribronchial lymphoid hyperplasia in lungs, multifocal and minimal cortical vacuolation in adrenals, dilated glands in stomach, sub-mucosal lymphoid hyperplasia in rectum and minimal presence of hyaline casts in kidneys. All these changes were of minimal severity and mostly solitary. These changes are commonly observed in rats of this age and were not considered to be test item related. Histopathological examination of the reproductive organs of male and female rats did not reveal any test item related morphological alterations.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Exposure of female rats to the test substance at and up to the dose levels of 1000 mg/kg bw/day did not have any adverse effect on the length and frequency of their oestrous cycle. Total 60 females were screened for normal oestrous cycle for 2 weeks prior to the treatment period. All females exhibited oestrous cycle of 4-5 days. Stages of 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.
The group mean values of the number of oestrous cycles during the two weeks before the mating period was 2.0, 2.0 and 2.2 among the females exposed to test item at the dose levels of 250, 500 and 1000 mg/kg bw/day respectively. These values were found to be comparable to that (2.3) in the control group.
The length of oestrus cycle was measured in days starting from detection of oestrous stage of the cycle till the oestrous stage of the subsequent cycle. The values of group mean length, in days, of the oestrous cycle occurring for two weeks before the mating period were 5.53, 5.79 and 4.94 among the females exposed to test item at the dose levels of 250, 500 and 1000 mg/kg bw/day respectively and were found to be comparable to that (5.57 days) of the control group. Thus the test item did not have any adverse effect on the oestrous cycle during the treatment period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The pregnancy rate was 66.7% in control and 60.0%, 60.0%, 53.3% in 250, 500 and 1000 mg/kg bw/day dose groups respectively. There was no test item related adverse effect on pregnancy rate. The mean gestational length (duration of pregnancy in days) computed for groups G1 to G4 for all dams was 22.9 ± 1.7 days, 22.8 ± 1.8 days, 23.1 ± 1.8 days and 22.8 ± 1.9 days respectively. The values do not differ from each other significantly (p>0.05).
No abortion/premature deliveries were observed in any of the dose levels during the study period.
The mean live birth index was 100% in control whereas it was 97.2%, 100% and 87.5% in G2, G3 and G4 respectively. This live birth index (number of live foetus on day 0 / number of foetuses born X 100) measures the female’s ability to maintain pregnancy, based on having delivered at least one live pup. The values do not differ from each other significantly (p>0.05). This indicated that the test item does not influence the live birth index.
There were no significant differences (p>0.05) in the group mean values of number of implantations, live, dead and total, observed in control group and groups treated with the test substance. This indicated that the test item did not influence the implantation process.
The total number of implants in control group dams was 82 in control whereas it was 84, 75 and 94 in treated group G2, G3 and G4 respectively. The group mean values of the total number of implants per female were 8.2 (Control), 9.7 (G2), 8.3 (G3) and 11.8 (G4).
Post-implantation loss: The group mean values of percent post implantation loss was 4.0 (Control), 4.2 (G2), 12.5 (G3) and 2.9 (G4).

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed up to and including the highest dose level tested.

2

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect observed up to and including the highest dose level tested.

3

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effect observed at this dose level.

4

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment with the test substance did not induce any abnormal clinical signs, indicative of systemic toxicity, in offspring during lactation period.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Treatment of dams with the test substance did not have any adverse effect on the survival of the
offspring during lactation period. The incidences of litters with still-born pups, pups found dead in
cage or cannibalized pups were very small and/or comparable across the treated and the control
groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment of dams with the test substance did not have any adverse effect on the offspring's body
weight gain during the lactation period.
The values of mean litter body weights and mean litter body weight gain for the offspring of female rats treated with the test substance did not differ significantly (p>0.05) from those of the concurrent
control group females during the postnatal lactation period except in G2 and G4. However, at two
time points on day 2 and day 13 postnatal lactation period, there was a significantly lower (p<0.05) body weight gain. The same group pups had gained weight at all other time points up to 13 days which was comparable statistically to that of control animals at all time points and hence of no toxicological significance.

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:

no effects observed

Description (incidence and severity):

The test item, at and up to the dose level of 1000 mg/kg bw/day, did not induce any changes in the total T4 on day 4 after birth and also on day 13 of lactation.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Treatment of dams with the test substance did not have any adverse effect on the anogenital distance for male and female pups as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the postnatal day (PND) 0 to PND 4. Only significant lowering (p<0.05) of anogenital distance was observed in male and female pups from G2
which was considered as incidental and not test item related.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

The number of nipples / areolae in male pups was counted on PND 12 among all male pups from control and treated groups. One male each from control, G2 and G4 were observed with retention of
nipple which was considered as incidental. Thus, the test item did not influence the nipple retention in male pups at postnatal day 12.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

On lactation day 13, average absolute weights of thyroid gland from pups of treatment groups i.e.
G2 (0.011g), G3 (0.008 g) and G4 (0.009 g) were found to be comparable with the control group (0.008 g).

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

offspring developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed up to and including the highest dose tested.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Summary data on body weight in males during the premating and mating period

Group	Dose (mg/kg bw/day)	Study Day	Treatment Period
			0	7	14	21	28
Vehicle control: corn oil
G1 5 mL/kg bw/day		Mean	489.3	515.2	530.7	541.5	550.4
		± S. D.	12.01	18.41	17.46	22.04	26.61
		n	10	10	10	10	10
Test item
G2	250	Mean	488.9	513.8	532.7	542.8	542.5
		± S. D.	13.67	20.46	21.41	16.88	16.93
		n	10	10	10	10	10
G3	500	Mean	488.0	518.2	530.7	533.1	539.4
		± S. D.	8.45	9.50	13.29	13.36	13.72
		n	10	10	10	10	10
G4	1000	Mean	489.00	513.30	515.00	491.50*	479.30*
		± S. D.	17.31	19.71	35.40	49.14	51.62
		n	10	10	10	10	10

* p<0.05 compared to control group

 

Table 2: Summary data on body weight gain in males during premating and mating period

Group	Dose (mg/kg bw/day)		Study Days
			0-7	7-14	14-21	21-28	0-28
Vehicle control: corn oil
G1  5 mL/kg bw/day		Mean	5.28	3.03	2.03	1.64	12.52
		± S. D.	1.99	1.35	2.17	2.30	5.35
		n	10	10	10	10	10
Test item
G2	250	Mean	5.08	3.68	1.94	-0.05	10.98
		± S. D.	2.39	1.34	1.74	1.48	2.59
		n	10	10	10	10	10
G3	500	Mean	6.2	2.41	0.46	1.19	10.54
		± S. D.	1.55	1.39	1.37	1.01	2.29
		n	10	10	10	10	10
G4	1000	Mean	4.97	0.32	-4.27*	-2.19	-1.76*
		± S. D.	1.86	5.47	10.13	9.40	11.98
		n	10	10	10	10	10

*: p<0.05 compared to control group

 

Table 3: Summary data for body weight gain in females during the premating period

Group	Dose (mg/kg bw/day)	Study Days
			0-7	7-14	0-14
Vehicle control: corn oil
G1		Mean	-0.23	2.21	1.97
		± S. D.	1.42	2.10	2.34
		n	15	15	15
Test item
G2	250	Mean	0.97	3.05	4.05
		± S. D.	1.66	1.56	2.05
		n	15	15	15
G3	500	Mean	4.46*	3.50	4.46
		± S. D.	3.97	1.57	3.97
		n	15	15	15
G4	1000	Mean	-0.69	3.18	2.49
		± S. D.	3.16	2.80	4.56
		n	15	15	15

* p<0.05 compared to control group

Applicant's summary and conclusion

Conclusions:

In the present combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, which was conducted according to OECD 422 and compliant with GLP, Wistar rats were exposed to 0 (control), 250, 500 and 100 mg/kg bw/day test substance via daily oral gavage, covering pre-mating, mating, gestation, lactation periods, up to day 13 post-partum (females) or day 28 after mating (males).
There were no adverse effects on developmental and reproductive parameters, neither in the parents, nor in the offspring. A decrease in body weight and body weight gain was observed in males during premating and mating period at the top dose of 1000 mg/kg bw/day. No other adverse effects were observed. Therefore, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of the test substance in Wistar rats, for systemic toxicity following oral administration for a period of four weeks for male rats was found to be 500 mg/kg bw/day and for a period of 50 to 60 days for females was found to be 1000 mg/kg bw/day. The No-Observed-Adverse-Effect-Level (NOAEL) of EC 453-490-7 in Wistar rats for reproductive and developmental toxicity was found to be 1000 mg/kg bw/day.