Dimethyl(octyl)amine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-31 to 2017-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Inadvertently, female animal no. 71 was not dosed and observed for clinical signs and mortality on post natal day 7. Inadvertently female as well as male pups were used for blood sampling for the analysis of T4 hormones.

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl(octyl)amine

EC Number:

230-939-3

EC Name:

Dimethyl(octyl)amine

Cas Number:

7378-99-6

Molecular formula:

C10H23N

IUPAC Name:

dimethyl(octyl)amine

Test material form:

liquid

Details on test material:

Name: AD-1
Batch number: 13602808
Appearance/colour: liquid/clear
Chemical name: N,N-dimethyloctylamine
CAS number: 7378-99-6
Purity nominal: > 95% w/w
Content of a.i. analysed: not available
Molecular weight: 157.3 g/mol
Relative density: 0.765
Risk symbol(s): Danger
Date of certificate: Not available
Expiry date: n/a
Storage conditions: ambient, dark, dry

Specific details on test material used for the study:

Name: AD 1
Chemical Name: N, N-Dimethyloctylamine
CAS No.: 7378-99-6
Batch No. 13602808
Expiry Date: not applicable
Physical State at Room Temperature: liquid
Colour: clear
Volatility: volatile
Density: 0.765
Molecular Weight: 157.3
Active Components: > 95%
Structure Formula of Active Component: C8H17-N-(CH3)2
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 329 - 386 g (mean: 347.90 g, ± 20% = 278.32 – 417.48 g)
females: 203 - 250 g (mean: 225.33 g, ± 20% = 180.26 – 270.39 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). Acidification of the water applies to the drinking water for mice and rats only. The purpose of the acidification is to provide additional protection against microbial contamination. This is a standard veterinary practice at BSL Munich and should not be associated with any risk for long-term studies.
- Animals were housed in groups of 5 animals/sex/cage in IVC cages (type IV, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration. Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and females (10 females/ group) with regular estrous cycle (4-5 day cycle) was used in the study.
Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively.
Each animal was marked with its identification number by individual ear tattoo.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Remarks:

Manufacturer: Sigma-Aldrich; Batch No.: MKBQ9948V; Expiry Date: 06 July 2016 and 01 September 2016

Details on exposure:

The vehicle was selected based on the test item’s characteristics and testing guideline.
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes.
The test item formulation was prepared at least once every ten days based on available stability data (Eurofins Munich study no. 154456). The prepared formulation was stored at room temperature.
The vehicle was also used as control item.


The following doses were evaluated:
Control: 0 mg/kg/d
Low Dose: 30 mg/kg/d
Medium Dose: 75 mg/kg/d
High Dose: 150 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad iniectabilia using the same
volume as used for the dose group.


The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day ‘0’ of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis were made prior to initiation of dosing which included stability analysis and homogeneity investigation. Study pre start stability analysis included samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8°C), 10 day -20°C and freeze thaw stability (16 samples).
Prestart homogeneity investigation included samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples). As the test item formulation was proved to be a true solution the homogeneity investigation was not repeated in the samples collected during the study.
During the study samples were collected for the investigation of homogeneity and nominal substance concentration. Samples meant for homogeneity analysis were not analysed as the formulation was a true solution.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
All formulation samples collected during the study were analysed on the same day. Samples of study week 3 were stored at -15 to -35°C for one day prior to analysis. These samples were analysed at Eurofins Munich under the BSL study no. 154457.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

Frequency of treatment:

daily

Details on study schedule:

Arrival of the Test Item: 17 August 2015
Study Initiation Date: 31 May 2016
Amendment to Study Plan: 06 June 2016
2nd Amendment to Study Plan: 23 June 2016
3rd Amendment to Study Plan: 05 October 2016
4th Amendment to Study Plan: 26 June 2017
Delivery of Animals: 02 June 2016
Acclimatisation Period: 02 June 2016 – 07 June 2016
Experimental Starting Date: 07June 2016
Treatment Period: 21 June 2016 –25 August 2016
Necropsies: 19 July 2016, 20 July 2016, 31 July 2016, 01 August 2016, 03 August 2016,
09 August 2016 – 14 August 2016, 21 August 2016 and 26 August 2016
Experimental Completion Date: 26 August 2016
Completion Date of Delegated Phase (Histopathology): 07 August 2017
Completion Date of Delegated Phase (Formulation Analysis): 29 September 2016

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

medium dose

Doses / concentrations 3

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

104 animals (42 males and 62 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study.

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made once a day, at the same time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment from all animals and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests:
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups. All animals were weighed at termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals.

Blood Coagulation
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment starts to select the females with regular cyclicity for the study. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight). The number of nipples / areolae in male pups were counted on PND 12.

Postmortem examinations (parental animals):

Pathology
The males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using an anesthesia (e.g. ketamine/xylazin). All surviving pups were killed by cervical dislocation on PND 13 (a day before adult female sacrifice).
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Non-pregnant females were sacrificed on study day 26 from the day of mating or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid gland and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s solution for 24 hours and then transferred to 70% ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The wet weight of the organs (testes, epididymides, prostate, seminal vesicles and coagulating glands, Cowper’s gland, thyroid gland (from 1 pup/sex/litter/group and from all adult males and females) was weighed after fixation, liver, kidneys, adrenal, pituitary gland, levator ani + bulbocavernosus muscle complex, glans penis, uterus with cervix, ovaries, thymus, spleen, brain, heart) of five randomly selected male and female animals (only lactating females were evaluated) from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of the animal found dead were not recorded.
Reproductive organs were weighed from all animals.
Thyroid gland from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. The thyroid gland weight was measured after fixation.

The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum,
epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs,
lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas,
parathyroid glands, pituitary) from the five randomly selected male and female animals were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
From the rest male and females (not randomly selected for full histopathology) only testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were collected and preserved.
The animal found dead was subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid gland from 1 pup/sex/litter/ group sacrificed on PND 13 and non selected adult animals was preserved for the potential histopathological examination. The thyroid gland weight was measured after fixation.


Histopathology
A full histopathology was carried out on the preserved organs and tissues (adrenal glands, all gross lesions, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole
rectum, sciatic nerve, skeletal muscle, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes,
thymus, thyroid gland, trachea, urinary bladder, uterus with cervix and vagina) of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Histopathological examination of thyroid gland from pups and from the remaining non-selected adult animals examination was not deemed necessary.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity. 
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals.
Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
Due to test item-related morphologic changes detected in stomach of high dose females, this organ was examined also from five randomly selected females from the low and medium dose groups.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. 
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Postmortem examinations (offspring):

Dead pups and pups sacrificed on PND 4 or 13 were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals of the main groups using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

The copulation, fertility and the delivery indices were calculated.

Offspring viability indices:

The viability index (%) from PND 0 to PND 4 ((No. of live offspring at day 4 / No. of live offspring at birth) X 100) as well as
the viability index (%) from PND 4 to PND 13 ((No. of live offspring at day 13 / No. of live offspring at day 4) X 100) were calculated separately.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males, there were no clinical symptoms up to the LD group. In the MD group, crust on ear (1/10), red nasal discharge (1/10) and slight salivation (1/10) were observed. In the HD group, moving the bedding (10/10) and salivation (slight (9/10), moderate (10/10) and severe (3/10)) were observed. The clinical symptoms in the MD group were transient in nature. In the HD group, moving the bedding and salivation were seen post the dose administration on all days. These findings were considered treatment related.
In females, there were no clinical symptoms up to the LD group. In the MD group, moving the bedding (10/10), slight salivation (9/10) and moderate salivation (2/10) were observed. In the HD group, there were symptoms of moving the bedding (10/10), slight piloerection (2/10), slight to moderate salivation (10/10) and severe salivation ( 3/10).
The clinical symptoms in the MD group were transient in nature. In the HD group, moving the bedding and salivation were observed on all days post dose administration. The slight piloerection in 2 females of the HD group were transient. These findings were considered treatment related.
The weekly detailed clinical examination showed no significant differences in test-item groups compared to the corresponding control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred due to test-item toxicity. However, 1 female (no. 77) died post dose administration on PND 2 due to gavaging error.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant changes observed in mean body weights or mean body weight gains in males during premating, mating or at sacrifice, or in females during premating, gestation or lactation. There was negative weight gain in LD, MD and HD females between lactation days 0-4 that coincided with PND 0-4. However, there was no statistically significant differences between the control and test item groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, treatment with AD 1 had no adverse effect on food consumption. In females of the LD, MD and HD groups a dose related decrease in food consumption compared to the control group was observed during the premating period. The food consumption was statistically significantly lower in the week-1 of gestation (GD 0-7) in HD group. Food consumption was also statistically significantly lower during 2nd and last week of gestation in LD and MD groups. During the lactation period, lower food consumption was observed in LD and HD groups, but without attaining the statistical significance and without the dose response relation. The lower food consumption in females could be attributed to a local irritation in forestomach by the test item as observed microscopically in HD group. These findings, in the absence of adverse effects on body weight development were not considered an adverse effect of test-item treatment.

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):

The haematology parameters of both genders were not affected in test-item groups compared to the corresponding control group.
The coagulation parameters were also not affected in test-item groups of both sexes compared to the control group. However, the mean PT values were very slightly but statistically significantly higher in male MD group (22.44 sec) compared to control (20.38 sec). This finding was not dose response related and was not considered to be an adverse effect of test item treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The clinical biochemistry parameters of both males and females were not affected in test-item groups compared to the corresponding controls. The mean values of test-item groups were not statistically different from the corresponding control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The neurological assessment revealed no significant differences in functional neurobehavioural parameters between the controls and test-test item treated groups.
The mean of the number of urination incidences (count) in male animals of the HD group was lower (0.2 counts) compared to control (0.6 counts), but did not differ statistically significantly. Therefore, the finding was not considered to be an adverse effect of test-item treatment. Very slight but statistically significant differences in body temperature between HD males and respective controls or urination between MD females and respective controls are not assumed to be toxicologically relevant.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The treatment with the test item AD 1 induced minor degrees of focal acute inflammation, focal parakeratosis and/or diffuse hyperkeratosis in the forestomach of three high dose females. These findings were considered to be due to a local irritation by the test item when administered orally.
The test item AD 1 produced no morphological evidence of toxicological properties in the reproductive organs and tissues examined.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. All findings recorded were within the range of normal background alterations.
Based on morphological findings recorded, a histopathological NOEL (No Observed Effect Level) for systemic and reproductive toxicity could be established at 150 mg/kg bw.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone (T4) Analysis
In adult males, the serum T4 hormone level was slightly lower in the MD (57.51 nmoL/L) and HD (59.94 nmoL/L) groups compared to the corresponding control group (63.54 nmoL/L). Because there was no statistically significant difference and there was no dose response related changes, the finding was not considered to be an adverse effect of treatment with AD 1.
In pups, the serum T4 hormone level was higher in the LD (70.02 nmoL/L), MD (94.61 nmoL/L) and HD (87.07 nmoL/L) groups compared to the corresponding control group (65.68 nmoL/L). The values in the test-item groups were not statistically significantly different and, moreover, there was no dose response relation. Thus, these differences are not considered to be an adverse effect. There were no effects on thyroid weights of adults and pups in the test- item treated groups and also there were no histopathological findings in the thyroid gland of males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of AD 1 on estrous cyclicity of females of test-item treated and control groups examined during the premating period of the study.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Precoital Interval and Duration of Gestation
There were no effects of AD 1 on the duration of precoital interval and the gestation.

Pre- and Post-Natal Data
There were no statistically significant effects on post natal parameters including corpora lutea, implantation sites and number of live pups at PND 0. There was no dose related increase in percent pre-implantation loss in test-item treated groups compared to the corresponding control group. The percent post-implantation loss was higher in the HD (17.94%) group than in the corresponding control group (7.37%). However, there was no dose response effect and no statistically significant difference between test-item treated groups and the corresponding control group.

Reproductive Indices P0
The copulation, fertility and the delivery indices were not affected by treatment with AD 1.
The mean viability index of pups from PND 0 to 4 was markedly lower in the HD group, which was due to two females (no. 71 and 79), wherein 3 pups of dam 71 and 1 pup of dam 79 were found dead on PND 1. Additionally, 1 pup of dam 79 was found dead on PND 3. The pups of dam 71 were hypothermic (cold) and the pups of dam 79 showed no indication of suckling. The parent females did not show any sign of distress or adverse toxicity due to test-item toxicity and therefore, the maternal toxicity was discounted. The findings were attributed to developmental toxicity.
The viability index from PND 4 to 13 was not affected in test-item treated groups compared to the corresponding control group.

Litter Data
Though there were lower mean number of live pups and lower mean number of female pups in the HD group on PND 0 and 4 compared to the corresponding control group, there were no statistically significant or dose-response-related effects on live births, stillbirths and runts on PND 0 and total number of pups, number of male or female pups, or the sex ratio of pups on PND 0 , 4, 13. Though not achieving statistical significance, there was a higher number of stillbirths and pup mortality in the HD group. These findings were related to test item treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Remarks:

for systemic and reproductive toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Based on morphological findings recorded, a histological NOEL (no Observed Effect Level) for systemic and reproductive toxicity could be established at 150 mg/kg body weight/ day.

2

Key result

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Based on the available results, the NOAEL for the systemic toxicity of male and female wistar rats was 150 mg/kg body weight/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a malformation “malpositioned limb” in one pup of the HD group (dam 80, pup 5). This malformation was in a single isolated pup of female no. 80 of the HD group and therefore, was considered incidental. Furthermore, in the control group a few pups showed no indication of suckling (dam no. 49- pup no. 1,3, 5) and no hair coat at necropsy (dam no. 50- pup No. 1, 3, 5 ,6, 7).
In the LD group, there was a scab on the abdomen (dam no. 56- pup no. 4), a haematoma on the abdomen (dam no. 58- pup no. 5), a small pup (dam no. 59- pup no. 7), no indication of suckling (dam no. 59- pup no. 10) and an absent hair coat (dam no. 59- pup no. 1- 8, 10,11).
The pups in the MD group had a hematoma on neck (dam no. 63- pup no. 3), small pups (dam no. 69- pup no. 11; dam 70- pup no. 1) and had no indication of suckling (dam no. 69- pup no. 11).
The findings in the pups of the HD group were: cold (dam no. 71- pup no. 1-11), small pup (dam 74- pup no. 4), no indication of suckling (dam 79- pup no. 5-6), no indication of suckling and cold (dam 80- pup no. 1- 4), small and skinny (dam no. 80- pup no. 3) and hindpaw malpositioned (dam 80- pup no. 5).
The findings scab, hematoma, small/thin pups and absent hair coat were in few pups of isolated females of control and test-item treated groups and were considered spontaneous in nature.
The findings “no indication of suckling” and/ or “hypothermic (cold)” were seen in the control group (1/10 females) and test-item groups (LD 1/10 females, MD 1/9 females, HD 3/9 females). The findings in the HD group only were considered to be an effect of treatment with AD 1 because of the increased incidences and were a sign of developmental toxicity.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No statistically significant effects were observed in mean pup survival, though pup mortality from PND 0 to PND 4 was higher in the HD group (15.91%) compared to corresponding control (4%). The pup mortality was considered to be an effect of test-item treatment.
The pup survival from PND 4 to PND 13 was not affected in test-item treated groups compared to the control.

The mean viability index of pups from PND 0 to 4 was markedly lower in the HD group, which was due to two females (no. 71 and 79), wherein 3 pups of dam 71 and 1 pup of dam 79 were found dead on PND 1. Additionally, 1 pup of dam 79 was found dead on PND 3. The pups of dam 71 were hypothermic (cold) and the pups of dam 79 showed no indication of suckling. The parent females did not show any sign of distress or adverse toxicity due to test-item toxicity and therefore, the maternal toxicity was discounted. The findings were attributed to developmental toxicity.
The viability index from PND 4 to 13 was not affected in test-item treated groups compared to the corresponding control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On PND 0, the pup mean weight in the HD group was slightly lower (6.06 g) compared to the control group (6.53 g). The pup mean weight in the HD group was markedly lower on PND 4 (9.57 g) and on PND 13 (25.17 g) compared to the corresponding control group (11.74 and 28.80, respectively). This coincided with negative mean body weight gains (-9.88 g) in HD dams during this same interval. However, these changes were not statistically significantly different.
Further, mean total litter weight and mean total female litter weight were lower in the LD and HD groups and the mean total male litter weight was lower in the HD group than in the control group on PND 0, 4 and 13 without attaining the statistical significance and also no dose response relationship. But considering the markedly lower mean litter weights in the HD group, the finding was attributed to higher pup mortality in HD group during the lactation period. Therefore, the finding was considered to be an effect of test-item treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Anogenital Distance and Nipple Retention
On PND 0, the pup mean weight in the HD group was slightly lower (6.06 g) compared to the control group (6.53 g). The pup mean weight in the HD group was markedly lower on PND 4 (9.57 g) and on PND 13 (25.17 g) compared to the corresponding control group (11.74 and 28.80, respectively). This coincided with negative mean body weight gains (-9.88 g) in HD dams during this same interval. However, these changes were not statistically significantly different.
Further, mean total litter weight and mean total female litter weight were lower in the LD and HD groups and the mean total male litter weight was lower in the HD group than in the control group on PND 0, 4 and 13 without attaining the statistical significance and also no dose response relationship. But considering the markedly lower mean litter weights in the HD group, the finding was attributed to higher pup mortality in HD group during the lactation period. Therefore, the finding was considered to be an effect of test-item treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

In pups, the weight of thyroid gland (weighed post fixation) of both male and female pups killed on PND 13 was not affected in the test-item groups. The mean values in the test-item groups were not statistically different to the corresponding control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see also clinical findings / gross external findings F1

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see also clinical findings / gross external findings F1

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

for the reproductive toxicity

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

developmental neurotoxicity

Remarks on result:

other:

Remarks:

The NOAEL for the reproductive toxicity was 150 mg/kg body weight.

2

Key result

Dose descriptor:

NOAEL

Remarks:

for the developmental toxicity

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

developmental neurotoxicity

Remarks on result:

other:

Remarks:

The NOAEL for the developmental toxicity was 30 mg/kg body weight.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with AD 1 in male and female Wistar rats with dose levels of 30, 75, and 150 mg/kg body weight/ day the following conclusions can be made:
At ≥ 75 mg/kg body weight/ day, the clinical symptoms of toxicity (slight salivation, moving the bedding) were observed. Non significant effects on MD animals were observed: negative mean body weight (females on LD 0-4); reduced mean food consumption (females from GD 7-14 and GD 14-21) and increased relative anogenital distance in female pups.
At 150 mg/kg body weight/ day, the following were reduced, indicating a treatment-related effect: mean number of live pups and mean number of female pups (PND 0 and 4); pup mean weight (PND 0 and PND 4); mean total litter weight, mean total male litter weight and mean total female litter weight; mean viability index of pups from PND 0 to 4; pup survival.
Also at 150 mg/kg body weight/ day, the following were increased, indicating a treatment-related effect: stillbirths and pup mortality; increased relative anogenital distance in female pups; incidence of “no indication of suckling” and/or “hypothermia”; the incidences of nipple retention, though not significantly different than controls.
Based on morphological findings recorded, a histological NOEL (no Observed Effect Level) for systemic and reproductive toxicity could be established at 150 mg/kg body weight/ day. Based on the available results, the NOAEL for the systemic toxicity of male and female wistar rats was 150 mg/kg body weight/day. The NOAEL for the reproductive toxicity was 150 mg/kg body weight. The NOAEL for the developmental toxicity was 30 mg/kg body weight.

Executive summary:

The aim of this study was to assess the possible health hazards likely to arise from repeated exposure and possible effects of AD 1 on male and female fertility and embryo-fetal development after repeated dose administration in Wistar rats.

The test-item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females / group) with regular estrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                  0   mg/ kg bw/ day

Low Dose:             30  mg/ kg bw/ day

Medium Dose:      75  mg/ kg bw/ day

High Dose:          150mg/ kg bw/ day

The test item formulation was preparedat least once every ten daysbased on the results obtained from stability investigations (study 154456). The test item formulation was stored at room temperature.

The test item was dissolved in corn oil and administered daily during the study. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 4 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. One animal died (no. 77, HD group) due to gavaging error and was examined macroscopically. At the conclusion of the test, the surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment from all animals and in the last week of treatment infive randomly selected males and femalesof each group.

After 14 days of treatment, male and female animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12.

From 2 pups/litter on day 4 after birth; from all dams and 2 pups /litter at termination on day 13 and from all adults males at termination blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and from the adultmales were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from adult females and day 4 pups were not deemed necessary. Pup blood was pooled by litter for thyroid hormone analysis.

The males were sacrificed after completion of the mating period on treatment days 29 (except male no. 36 sacrificed on day 30) and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26 from the day of evidence of mating.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natalday 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed in animals of the control and HD groups and in non pregnant female animals of the LD and MD groups. Furthermore, preserved tissues were also examined in the mating partners of the non-pregnant female LD and MD animals.Due to test item-related morphologic changes detected in stomach of high dose females, this organ was examined also from five randomly selected females from the LD and MD groups.

Summary Results

AD 1 in corn oil was stable for up to 12 days under all conditions (0h, 6 hours RT, 12 days RT, 12 days 2-8°C, 12 days -15 to -35°C). Formulations of AD 1 in corn oil were homogenous at concentrations of 5 and 50 mg/mL, as COV was below or equal to 10%. At both concentrations no phase separation was observed within 30 minutes of formulation preparation and were considered as true solutions. The concentration analysis of formulation samples collected during the main study was determined at three concentrations, 7.5 mg/mL, 18.75 mg/mL and 37.5 mg/mL in study weeks 1,3,5 and in the last week of the study. The mean recoveries observed in the LD, MD and HD groups were 100.9%, 100.7% and 103.1% of the nominal concentration, respectively.

There was no mortality due to test-item treatment in the study. However, 1 female (no. 77) died post dose administration on post natal day 2 due to gavaging error.

The clinical symptoms of salivation and moving the bedding were observed in all HD animals and most MD females. Piloerection was also observed in two HD females in the study. These clinical symptoms were considered treatment related.

The weekly detailed clinical examination showed no significant differences in test-item groups compared to the corresponding control group.The neurological assessment revealed no significant differences between the controls and test-test item groups. There were no significant changes observed in mean body weights or mean body weight gains in males during premating, mating or at sacrifice, or in females during premating, gestation or lactation. There was negative weight gain in LD, MD and HD females between lactation days 0-4 that coincided with PND 0-4 (coinciding with pup PND 0-4). There was a dose response-related decrease in mean food consumption in treated females that was statistically significant in HD females on GD 0-7 and in LD and MD females from GD 7-14 and GD 14-21. The lower food consumption in females could be attributed to a local irritation in forestomach by the test item as observed microscopically in HD group.

There was no effect of AD 1 on estrous cyclicity of females in the test-item groups.

The hematology, blood coagulation and clinical biochemistry parameters were not adversely affected in test-item treated animals compared to the corresponding control animals.

The organ weights were not adversely affected in males and females of test item groups compared to the control.

The treatment with the test item AD 1 induced minor degrees of focal acute inflammation, focal parakeratosis and/or diffuse hyperkeratosis in the forestomach of three high dose females. These findings were considered to be due to a local irritation by the test item when administered orally. The test item AD 1 produced no morphological evidence of toxicological properties in the reproductive organs and tissues examined.

There was no adverse effect on the serum level of T4 thyroid hormone in the adult males and in the pups killed on PND 13. There was no adverse effect on thyroid weight in pups killed on PND 13.Though there were lower mean number of live pups and lower mean number of female pups in the HD group on PND 0 and 4 compared to the corresponding control group, there were no statistically significant or dose-response-related effects on live births, stillbirths and runts on PND 0 and total number of pups, number of male or female pups, or the sex ratio of pups on PND 0 , 4, 13. Though not achieving statistical significance, there was a higher number of stillbirths and pup mortality in the HD group. The pup mean weight in the HD group was lower on PND 0 and PND 4, respectively compared to corresponding control. This coincided with negative mean body weight gains (-9.88 g) in HD dams during this same interval.

Mean total litter weight and mean total female litter weight were non significantly lower in the LD and HD groups and the mean total male litter weight was non significantly lower in the HD group than in the control group on PND 0, 4 and 13.

The percent post-implantation loss was higher in HD (17.95%) group than in the corresponding control (7.37%) although this difference did not achieve statistical significance.

There were no statistically significant effects on post natal parameters including corpora lutea, implantation sites, number of live pups at PND 0, or in % pre or post implantation loss. The mean viability index of pups from PND 0 to 4 was markedly lower in the HD group compared to the corresponding control. No statistically significant effects were observed in mean pup survival, though pup mortality from PND 0 to PND 4 was higher in the HD group (15.91%) compared to the corresponding controls (4 %).

There were no statistically significant effects on anogenital distance and relative anogenital distance in treated male pups. Nipple retention was, however, seen in statistically significantly higher number of male pups of the MD groups. Because there was no dose response relationship, the finding was not considered to be an adverse effect. Relative anogenital distance was significantly increased in MD and HD female pups, but the anogenital distance was not. The statistically significantly higherrelative anogenital distance was attributed to the possible androgenic effect of the test item.

In the pups of the control and/or test-item groups, there were findings of scab, hematoma, small/skinny pups and absent hair coat in few pups of isolated females and these were considered spontaneous in nature.

Prior to being found dead or subjected to terminal sacrifice, there was “no indication of suckling” and/or “hypothermia” in control (1/10 females) and test-item groups (LD 1/10 females, MD 1/9 females, HD 3/9 females). In the HD, these were considered adverse effects of treatment, because of the increased incidences.

Conclusion

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with AD 1 in male and female Wistar rats with dose levels of 30, 75, and 150 mg/kg body weight/ day the following conclusions can be made:

At ≥75 mg/kg body weight/ day, the clinical symptoms of toxicity (slight salivation, moving the bedding) were observed. Relative anogenital distance was significantly increased in female pups. Non significant effects on MD animals were negative mean body weight (females on LD 0-4) and reduced mean food consumption (females from GD 7-14 and GD 14-21).

At 150 mg/kg body weight/ day, the following were reduced, indicating a treatment-related effect: mean number of live pups and mean number of female pups (PND 0 and 4); pup mean weight (PND 0 and PND 4); mean total litter weight, mean total male litter weight and mean total female litter weight; mean viability index of pups from PND 0 to 4; pup survival.

Also at 150 mg/kg body weight/ day, the following were increased, indicating a treatment-related effect: stillbirths and pup mortality; increased relative anogenital distance in female pups; incidence of “no indication of suckling” and/or “hypothermia”; the incidences of nipple retention, though not significantly different than controls.

Based on morphological findings recorded, a histological NOEL (no Observed Effect Level) for systemic and reproductive toxicity could be established at 150 mg/kg body weight/ day. Based on the available results, the NOAEL for the systemic toxicity of male and female wistar rats was 150 mg/kg body weight/day. The NOAEL for the reproductive toxicity was 150 mg/kg body weight. The NOAEL for the developmental toxicity was 30 mg/kg body weight.