6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2017 - 25 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

see "Any other information" for details.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

EC Number:

201-618-5

EC Name:

6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

Cas Number:

85-60-9

Molecular formula:

C26H38O2

IUPAC Name:

2-tert-butyl-4-[1-(5-tert-butyl-4-hydroxy-2-methylphenyl)butyl]-5-methylphenol

Test material form:

solid

Details on test material:

Test Item Name: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
Synonym: LOWINOX® 44B25
Laboratory Master Schedule No. at FumoPrep Ltd.: 4070
Manufacturer: Addivant USA LLC
Chemical Name: 2-tert-butyl-4-[1-(5-tert-butyl-4-hydroxy-2-methylphenyl)butyl]-5-methylphenol
Batch/Lot Number: W4B5A0009
CAS Number: 85-60-9
Appearance: White solid
Manufacture: Date 23 Jan 2015
Expiry Date: 22 Jan 2019
Purity: Treated as 100%
Storage Condition: Controlled room temperature (15-25 °C, below 70 RH %)
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

No further information available.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar rats (CRL:WI(Han))

Details on test animals or test system and environmental conditions:

Source:
Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony

Justification of strain:
The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to the experience of the Test Facility with this strain in teratology studies.

Housing condition:
Standard laboratory conditions; individual housing

Number of animals:
98 female animals, 24-25 mated female animals/group, 4 groups (one control and 3 test item-treated groups); 21, 22, 20, 23 pregnant and evaluated female animals (with implantation sites at necropsy) per Control, Low, Mid and High dose groups, respectively; 60 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were used

Age of animals:
Young adult female rats, nulliparous and non-pregnant, at least 11 weeks old at mating.

Starting body weight:
183-236 g (the variation did not exceed ± 20% of the mean weight)

Acclimation period: at least 12 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian.

Cage type: Type II polycarbonate cages were used during mating and gestation period and Type III polycarbonate cages were used during the acclimatisation period

Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany were used in the study (Batch number: 03018170104, Expiry date: 04 January 2020).

Nesting: Arbocel crinklets natural nesting material produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) was used in the study (Batch number: 05072160415, Expiry date: 15 April 2019).

Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Temperature: 20.2-26.8°C (target: 22 ± 3°C)

Relative humidity: 28-65% (target: 30-70%)

Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Successfully mated animals were housed individually. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.

The temperature and relative humidity were monitored continuously and recorded twice on each day during the study. The bedding and nest building material were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Diet and water supply

The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum. The diet and drinking water were routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The standard content of the diet as provided by the supplier and the results of the diet analysis (Batch number: 285 17890, Expiry date: August 2017) are presented in Appendix 8.

The quality control analysis of the water is performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36, Hungary). Copies of the relevant Certificates of Analysis are retained in the Archives of CiToxLAB Hungary Ltd.

Preparation of the animals

Animal Identification

Adult animals were identified by temporary numbers written with indelible ink on the tail during the entire study. During necropsy and Caesarean section procedures each evaluated dam was given an additional number (evaluation number indicating group number), and cross-referenced with the numbers used during the in-life phase of the study.

The cages were marked with individual identity cards, with information about study code, sex, dose group, cage number, animal number, date of mating and caesarean section/necropsy date. Cages were arranged to minimise any possible effects due to cage placement.

The litters were identified at necropsy with litter numbers. The flasks used for fixation and all sheets used for recording data were identified only by the litter numbers until the end of foetal examinations (facilitating blind examination of foetuses).

The foetuses were identified during the Caesarean section with individual numbers according to their implantation sites. For visceral examination they were identified by digit-clipping; for skeletal examination the foetuses were identified by means of a water-proof plastic ribbon tied around their neck.

Mating Procedure

The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male : 1 female) until at least 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Randomisation

The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. A computer software (SPSS PC+4.0) was used to verify homogeneity/variation among/within groups.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks on MMAD:

Not applicable.

Details on exposure:

A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.

The control or test item dose formulations were administered to mated, sperm positive (assumed pregnant) female rats daily by oral gavage on a 7 days/week basis, approximately at similar times, from gestation day 6 (GD 6) to gestation day 19 (GD 19)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in the vehicle (Propylene glycol) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd.

Formulations were prepared prior to administration to the animals at the appropriate frequency (not more than 7 days before use).

Stability of the test item in the vehicle was assessed in the conditions employed on the study during the analytical method validation (CiToxLAB study code: 16/446-920AN [3]). In this study, test item formulation samples in the 10-250 mg/mL concentration range (using Propylene glycol as vehicle) were proven to be stable for at least 15 days when stored at room temperature (20±5oC).

Analysis of test item formulations for concentration and/or homogeneity was performed at the Test Site using a validated HPLC (High Pressure Liquid Chromatography) method [3]. Top, middle and bottom samples were taken from the test item formulations two times during the study (during the first week of the treatment and once on the second half of treatment). Samples were taken in duplicate (0.5 mL/each), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Formulation samples were kept at room temperature until shipment. Samples (both sets) were shipped as soon as possible after collection for concentration and/or homogeneity measurements to the Principal Investigator (PI). The number and date of shipments was agreed with the PI.

Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration.

Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) had to be less than 10%.

The formulation analysis was conducted under the control of the Principal Investigator in compliance with the Study Plan, and the relevant Standard Operation Procedures (SOPs) of the Test Site for analytical work. The results of the analysis are included in the study report as an appendix (in a form of a Phase Report, Phase Report code: FPBSTUDY-160-MEAS2).

Any samples not required for analysis were discarded following acceptance of the results of the formulation analysis by the Principal Investigator and Study Director.

Details on mating procedure:

Mating Procedure

The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male : 1 female) until at least 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Randomisation

The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. A computer software (SPSS PC+4.0) was used to verify homogeneity/variation among/within groups.

Duration of treatment / exposure:

Start of experiment: 25 April 2017 (first mating, when the females were at least 11 weeks old)
Start of treatment: 01 May 2017 (first Gestation Day 6, GD6)
End of treatment: 24 May 2017 (last Gestation Day 19, GD19)
End of experiment : 25 May 2017 (last necropsy day)

Frequency of treatment:

7 days/week

Duration of test:

30 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

The number of confirmed pregnant, evaluated dams was 21 in the Control, 22 in the Low (100 mg/kg bw/day), 20 in the Mid (300 mg/kg bw/day) and 23 in the High (1000 mg/kg bw/day) dose groups, respectively.

Control animals:

yes, concurrent vehicle

Details on study design:

See "any other information" below.
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day). Only one general clinical observation was made on the first day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observation (detailed) was made only once on necropsy days (in the morning).

Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.

Examinations

Maternal examinations:

The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

Ovaries and uterine content:

Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital (RELEASE®, 300 mg/mL sodium pentobarbital solution; Supplier: Wirtshaftsgenossenschaft Deutscher Tierärzte (Address: Siemensstrasse 14., D-30827 Garbsen, Germany), Batch number: 106075, Expiry date: July 2018) administered by intramuscular injection and followed by exsanguination was used for euthanasia.

The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. Due to a technical oversight, the uterus was not weighted for two animals with early embryonic deaths (#1521 in the Control, and #3520 in the Mid dose group).

The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the appearance of the anogenital distance.

Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination.
For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sannomiya mixture (a mixture of 920 mL concentrated isopropanol, 30 g sulfosalicylic acid an 50 mL acetic acid), then, after fixation, the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue - acetic acid – isopropanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.

All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.

No histopathology evaluation was performed in the study (as it was not required for interpretation).

Statistics:

Data were collected using the software PROVANTIS v.9 (maternal data, Caesarean section and necropsy data), or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd. (foetal evaluation data) then tabulated with PROVANTIS v.9 or Microsoft Office Excel 2010. Data were collected to provide information on parameters including:

Maternal Data:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day)
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD 20 (body weight minus gravid uterine weight) and corrected body weight gain GD 0-20 (body weight gain (GD 0-20) minus gravid uterine weight): mean ± S.D.
- Net body weight change (body weight gain during the treatment period (GD 6-20) minus gravid uterine weight): mean ± S.D.
- Gravid uterine weight: mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings, placenta findings

Indices:

Not reported.

Historical control data:

All paramaters where in accordance with the historical control data utilised by the laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality was recorded for any animals in the study.

No test item-related effects or systemic clinical signs were noted in the treated animals of the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) or High (1000 mg/kg bw/day) dose groups.

Vaginal discharge was observed on Day 14 and 15 in one non-pregnant, excluded High dose animal (#168), but this finding was not considered as a test item related effect.

Furthermore, thin fur / alopecia on some areas was recorded for two animals in the Control group (animal numbers #1506 and #1508) from Day 14 and day 19, respectively. These changes were considered to be incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was recorded for any animals in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related effect on body weight was observed in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day) when compared to control.

No statistically significant changes were observed in the mean body weights (Figure 1) of the Low, Mid or High dose groups when compared to control. Similarly, no statistically significant or biologically relevant differences were seen in the body weight gain or corrected body weight gain or net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value, with the exception of GD 6-8, where significantly higher body weight gain was measured in the Mid and High dose groups (p < 0.05, and p < 0.01, respectively). This finding was considered to be incidental, and not a Test Item related effect. Net body weight gain was significantly higher in all treated dose groups when compared to the Control. This increase was not considered to be a Test Item related adverse effect..

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant changes were observed in the mean daily food consumption of dams in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively) compared to the control value.

The food consumption of the Low, Mid and High dose groups was comparable with the Control group, the difference in the daily mean food consumption (calculated for the entire period of the study) was not larger than 10.2% in any dose groups when compared to the control.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related observations were recorded for any evaluated animals in the study.

Alopecia was detected for one animal at necropsy (#1506), this finding is in correlation with the findings recorded during the in life phase. Thick fur was detected for one animal (#1508).

A grey/yellow, firm mass, with a diameter approximately 20 mm was observed on the left ventral thoracic region of a Mid dose animal (#3515).

However, these findings were considered as incidental and not related to the test item administration.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Ninety-eight females (24 for the Control and Low, 25 for the Mid and High dose groups, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was 21 in the Control group, 22 in the Low dose group (100 mg/kg bw/day), 20 in the Mid dose group (300 mg/kg bw/day) and 23 in the High dose group (1000 mg/kg bw/day).

There was no statistically significant difference in foetal death in any test item treated groups compared to the control. Summary of intrauterine evaluation is shown in
Table 4.

The mean number of corpora lutea was comparable with the control in all test item treated groups. No significant differences were noted in preimplantation loss or number of implantations of the test item treated groups when compared to the control.

The early and the late embryonic loss values of the test item treated groups were comparable with control. The number of death foetuses were 1, 3, 1, and 2 in 1, 3, 1 and 1 litters for the Control, Low, Mid and High dose groups, respectively. There was no statistically significant difference in the postimplantation loss or total intrauterine mortality between the test item treated and control groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Remarks on result:

other: No effects noted.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant adverse effect of the test item was observed on the foetal parameters in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The total number of retarded foetuses (runts) as well as the number of affected litters was similar in all test item treated group as in the Control group, indicating no effect on this parameter

External malformations:

no effects observed

Description (incidence and severity):

No external variations or malformations were recorded in the study

Skeletal malformations:

no effects observed

Description (incidence and severity):

Based on the skeletal findings the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively). In the case of most skeletal variations (ossified sternebra (2 or less), dumbbell / asymmetrically ossified vertebras, wavy ribs, hyoid body, 2 or more unossified vertebra, and bipartite ossification of the vertebra), the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

Dumbbell shaped vertebrae were considered incidental findings based on the isolated occurrence (recorded only in one foetus), and was not related to the treatment.

The incidence of Carpal, ossified ≤ 2.5 was higher in the Control and Low dose groups than in the relevant HC data, however, there was no dose response (the Mid and High dose group did not show this variation). A similar case was observed for large fontanelle, and for unossified/incompletely ossified pubis, where the foetal or litter based incidence in the Low dose group was higher than the study control or relevant historical control, but there was no dose response (the Mid and Low dose groups showed the variation comparable manner to study control or they did not even contain the variation). Furthermore, only ossified sternebra (2 or less) showed statistical significante in the Mid dose group, where this variation did not occurred, thus this finding were considered as incidental and not being treatment related.

Visceral malformations:

no effects observed

Description (incidence and severity):

All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings.

In case of several visceral variations (short brachiocephalic trunk, and small renal papilla, and large adrenal gland), the foetal or litter based incidence in the test item treated groups was comparable with the study control and/or historical control values. The incidences were low for all variations, in all groups. The variations observed were considered as incidental and not related to the test item treatment.

In case of dilated renal pelvis and absent renal papilla variations, recorded parallel only in one foetus the Low dose group, no similar observation was recorded in the study control or historical control database. But based on the low absolute value (only one foetus was affected), it was considered as incidental finding, unrelated to test item administration.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

In conclusion, all of the foetal findings, in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental finding and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment. Based on these results the test item did not affect adversely the intrauterine development.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity, foetotoxicity, teratogenecity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Any other information on results incl. tables

Table 1:Summary of the dose formulation analysis

Formulation	03 May 2017		16 May 2017
	Mean measured concentration (mg/mL)	Relative to the nominal concentration (%)	Measured concentration (mg/mL)	Relative to the nominal concentration (%)
Control	not detectable	-	not detectable	-
20 mg/mL	20.19 ± 0.11	100.9	19.61±0.24	98.1
60 mg/mL	57.75 ± 0.46	96.2	57.96±0.53	96.6
200 mg/mL	186.90 ± 2.58	93.4	189.11±2.67	94.6

Note: Samples collected freshly on the days indicated in the header of the table and sent within 2 days to the Test Site. They were stored at 20±5°Cbefore the analysis for a maximum of 2 days. Analysis was performed on 04, and 18 May 2017 for the two occasions respectively.The LOQ was 8.4 µg/mL in the analytical sample, which was equal to 8.4 mg/mL concentration in the formulation.

  

Table 2:Selected body weight parameters

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	21	22	20	23
Body weight on GD20 (g)	301.8	309.0	309.0	310.4	NS
Body weight gain GD6-20 (g)	67.2	75.5	73.7	75.1	NS
Body weight gain GD0-20 (g)	91.3	99.2	98.1	98.4	NS
Corrected body weight on GD20 (g)	253.90	257.95	259.21	261.13	NS
Corrected body weight gain GD0-20 (g)	43.70	48.23	49.63	49.09	NS
Net body weight gain GD6-20 (g)	19.50	24.45*	25.00*	25.78*	U

Notes: Body weight data were rounded to one decimal place. Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

NS: Statistically not significant when compared to the vehicle control.

U = Mann-Whitney U - test Versus Control

* = p < 0.05

 

 

Table 3: Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of mated females	24	24	25	25
Pre-terminal death or euthanasia	0	0	0	0
Number of non-pregnant females	3	2	5	2
Number of females with ≤ 5 implantation sites	1	1	2	1
Number of evaluated females on GD20 (Caesarean section)	21	22	20	23

Note: Three females with ≤ 5 implantation sites and 100 % intrauterine mortality (#1521, #2522 and #3520) were included in the evaluation.

 

Table 4: Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	21	22	20	23
Mean number of corpora lutea	11.57	11.86	11.30	11.13	NS
Preimplantation loss, mean	1.67	1.95	1.70	1.30	NS
Preimplantation loss (%), mean	14.76	15.95	17.80	11.78	NS
Mean number of implantations	9.90	9.91	9.60	9.83	NS
Early embryonic loss, mean	0.71	0.14	0.35	0.52	NS
Early embryonic loss (%), mean	11.48	2.27	7.45	7.30	NS
Late embryonic loss, mean	0.24	0.09	0.05	0.13	NS
Late embryonic loss (%), mean	3.05	0.86	0.40	1.09	NS
Dead foetuses, mean	0.05	0.14	0.05	0.09	NS
Dead foetuses (%), mean	0.68	5.46	0.45	0.79	NS
Postimplantation loss, mean	1.00	0.36	0.45	0.74	NS
Postimplantation loss (%), mean	15.19	8.59	8.30	9.13	NS
Total intrauterine mortality, mean	2.67	2.32	2.15	2.04	NS
Total intrauterine mortality (%), mean	26.81	18.86	23.30	18.30	NS
Viable foetuses, mean	8.90	9.55	9.15	9.09	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

 

Table 5: Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of examined litters	21	22	20	23
Viable foetuses, mean	8.90	9.55	9.15	9.09	NS
Male foetuses, mean	4.90	4.71	4.79	5.00	NS
Female foetuses, mean	4.45	5.29	4.84	4.09	NS
Total number of foetuses	187	210**	183*	209	CH
Total number of male foetuses	98	99	91	115	NS
Total number of female foetuses	89	111	92	94	NS
Sex distribution (% of males / females)	56 / 44	46 / 54	47 / 53	55 / 45	NS
Mean foetal weight / litter (g)	2.735	2.705	2.872	2.829	NS
Number of foetuses with retarded body weight	1	1	0	0	NS
Number of affected litters (with runts)	1	1	0	0	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control, NA: Not applicable. CH: Chi²Test, * = p < 0.05, ** = p < 0.01

 

Table 6: Summary table of the external abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	20	21	19	23	670
Total number of examined foetuses	187	210	183	209	6889
Total number of intact (normal) foetuses	187	210	183	209	--
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	0 / 0	--
Total number of foetuses / litters with variation	0 / 0	0 / 0	0 / 0	0 / 0	--
External malformations
No external malformation was recorded in the study.
External variations
No external variation was recorded in the study.

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

No statistically significant differences were noted when compared to the control group.

 

Table 7: Summary table of the visceral abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	19	21	19	23	670
Total number of examined foetuses	93	107	89	105	3450
Total number of intact (normal) foetuses	91	106	86	103	--
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	0 / 0	--
Total number of foetuses / litters with variation	2 / 2	1/ 1	3 / 3	2 / 2	--

 Notes:Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

No statistically significant differences were noted when compared to the control group.

  

Table 8: Details of the visceral abnormalities

Parameter			Dose (mg/kg bw/day)				HC data
			Control	100	300	1000
Total number of examined litters			19	21	19	23	670
Total number of examined foetuses			93	107	89	105	3450
Visceral malformations
No visceral malformation was recorded in the study.
Visceral variations
Brachiocephalic trunk, short	Litter incidence	n	2	0	2	0	45
		%	10.5	0.0	10.5	0.0	6.7
	Foetal incidence	n	2	0	2	0	53
		%	2.151	0.000	2.247	0.000	1.536
Renal papilla, absent	Litter incidence	n	0	1	0	0	--
		%	0.0	4.8	0.0	0.0	--
	Foetal incidence	n	0	1	0	0	--
		%	0.000	0.935	0.000	0.000	--
Renal pelvis, dilated	Litter incidence	n	0	1	0	0	--
		%	0.0	4.8	0.0	0.0	--
	Foetal incidence	n	0	1	0	0	--
		%	0.000	0.935	0.000	0.000	--
Renal papilla, small	Litter incidence	n	0	0	1	1	20
		%	0.0	0.0	5.3	4.3	3.0
	Foetal incidence	n	0	0	1	1	22
		%	0.000	0.000	1.124	0.952	0.638
Adrenal gland large	Litter incidence	n	0	0	0	1	1
		%	0.0	0.0	0.0	4.3	0.1
	Foetal incidence	n	0	0	0	1	1
		%	0.000	0.000	0.000	0.952	0.029

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted compared to the control group

 

 Table 9: Summary table of the skeletal abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	20	21	18	23	670
Total number of examined foetuses	94	103	94	104	3435
Total number of intact (normal) foetuses	81	87	88	96	--
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	0 / 0	--
Total number of foetuses / litters with variation	13 / 8	16 / 11	6 / 5	8 / 4	--

  Notes:Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

CH: Chi²test, *: p<0.05, **: p<0.01 compared to control group

 

 

Table 10: Details of the skeletal abnormalities

Parameter			Dose (mg/kg bw/day)				HC data
			Control	100	300	1000
Total number of examined litters			20	21	18	23	670
Total number of examined foetuses			94	103	94	104	3435
Skeletal malformations
No skeletal malformation was recorded in the study.
Skeletal variations
Skull, Fontanelle, large	Litter incidence	n	1	2	0	0	3
		%	5.0	9.5	0.0	0.0	0.5
	Foetal incidence	n	2	3	0	0	3
		%	2.128	2.913	0.000	0.000	0.087
Skull, Hyoid body, unossified	Litter incidence	n	2	1	0	0	--
		%	10.0	4.8	0.0	0.0	--
	Foetal incidence	n	2	1	0	0	--
		%	2.128	0.971	0.000	0.000	--
Ossified Sternebra (2 or less) #	Litter incidence	n	6	7	0	3	48
		%	30.0	33.3	0.0	13.0	7.2
	Foetal incidence	n	6	10	0*	5	57
		%	6.383	9.709	0.000	4.808	1.659
Rib, wavy, marked	Litter incidence	n	2	2	2	2	198
		%	10.0	9.5	11.1	8.7	29.6
	Foetal incidence	n	3	2	2	2	311
		%	3.192	1.92	2.128	1.923	9.054
Vertebra, dumbbell or asymmetric ossification (2 or more)	Litter incidence	n	1	2	0	0	85
		%	5.0	9.5	0.0	0.0	12.7
	Foetal incidence	n	1	2	0	0	92
		%	1.064	1.942	0.000	0.000	2.678

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

#: Ossified sternebra (3 or less) is recorded in the historical control database

No statistically significant differences were noted compared to the control group.

 

Table 10: Details of the skeletal abnormalities (continued)

Parameter			Dose (mg/kg bw/day)				HC data
			Control	100	300	1000
Total number of examined litters			20	21	18	23	670
Total number of examined foetuses			94	103	94	104	3435
Skeletal variations
Vertebra, sacral 2 or more unossified	Litter incidence	n	2	2	1	1	--
		%	10.0	9.5	5.6	4.3	--
	Foetal incidence	n	2	2	1	3	--
		%	2.128	1.942	1.064	2.885	--
Vertebra, dumbbell shaped	Litter incidence	n	0	0	1	0	9
		%	0.0	0.0	5.6	0.0	1.3
	Foetal incidence	n	0	0	1	0	9
		%	0.000	0.000	1.064	0.000	0.262
Vertebra, bipartite ossification	Litter incidence	n	0	0	1	1	27
		%	0.0	0.0	5.6	4.3	4.0
	Foetal incidence	n	0	0	1	1	27
		%	0.000	0.000	1.064	0.962	0.786
Pubis unossified or incomplete ossification	Litter incidence	n	3	2	2	0	6
		%	15.0	9.52	11.1	0.0	0.9
	Foetal incidence	n	3	3	2	0	6
		%	3.192	2.913	2.128	0.000	0.175
Carpal, ossified ≤ 2.5	Litter incidence	n	1	2	0	0	5
		%	5.0	9.5	0.0	0.0	0.7
	Foetal incidence	n	1	2	0	0	5
		%	1.064	1.942	0.000	0.000	0.146

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted when compared to the negative (vehicle) control.

 

Applicant's summary and conclusion

Conclusions:

In conclusion, LOWINOX® 44B25, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19), was associated with the following findings:

There were no mortalities or clinical signs in the study related to the test item treatment.

There was no test item related adverse effect on maternal body weight / body weight gain, maternal corrected body weight / body weight gain, and on food intake in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

There were no toxicologically significant differences, or test item related-changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean. No test item related effect was observed on the mean foetal body weight per litter.

No remarkable internal or external observations related to test item treatment were recorded for any pregnant animals during necropsy. No remarkable abnormalities were observed on the placentas in any examined groups.

There were no statistically significant effects of the test item on external, visceral and/or skeletal development of foetuses in the study. No malformations were observed in the study.

In this study, from the observations made in the dams and their foetuses, no signs of maternal toxicity were noted and there were no toxicologically relevant adverse effect on embryos or foetuses in any test item treated groups. The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 1000 mg/kg bw/day
NOAELembryotoxicity: 1000 mg/kg bw/day
NOAELfoetotoxicity: 1000 mg/kg bw/day
NOAELteratogenecity: 1000 mg/kg bw/day

Executive summary:

In conclusion, LOWINOX® 44B25,when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19), was associated with the following findings:

 

There were no mortalities or clinical signs in the study related to the test item treatment.

 

There was no test item related adverse effect on maternal body weight / body weight gain, maternal corrected body weight / body weight gain, and on food intakein the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

 

There were no toxicologically significant differences, or test item related-changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

 

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.No test item related effect was observed on the mean foetal body weight per litter.

 

No remarkable internal or external observations related to test item treatment were recorded for any pregnant animals duringnecropsy. No remarkable abnormalities were observed on the placentas in any examined groups.

 

There were no statistically significant effects of the test item on external, visceral and/or skeletal development of foetuses in the study. No malformations were observed in the study.

 

In this study, from the observations made in the dams and their foetuses, no signs of maternal toxicity were noted and there were no toxicologically relevant adverse effect on embryos or foetuses in any test item treated groups. The following no-observed-adverse-effect (NOAEL) levels were derived:

 

NOAEL_{maternal toxicity}:    1000 mg/kg bw/day

NOAEL_{embryotoxicity}:       1000 mg/kg bw/day

NOAEL_{foetotoxicity}:    1000 mg/kg bw/day

NOAEL_{teratogenecity}:   1000 mg/kg bw/day