Registration Dossier

Toxicological information

Toxicity to reproduction

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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:
2012-07-16 to 2013-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: Males and non-pregnant nulliparous females
Age at the beginning of the study: 10-11 weeks old
Body weight at the beginning of the study: interval within ± 20% of the mean weight.

The range of the body weight was:
Females: 172-203g, (mean: 190.15 ± 20%= 38.03 g)
Males: 285-318g, (mean: 300.03 g, ± 20%= 60.01 g)
Number of animals: 10 Males and 10 females per group
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 are bred for experimental purposes.


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 (lot no. 0939)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 030512)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- 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.
Before the first administration all animals 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.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: Sesame oil
Details on exposure:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle (sesame oil) was added to give the appropriate final concentration of the test item. The formulation vials were placed on Vortex machine for short period to ensure proper homogenistation of the formulation.

The vehicle was selected as suggested by the sponsor and on the basis of the test item’s characteristics.

The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations.

The following doses were evaluated:
Control: 0 mg/kg body weight/day
Low Dose: 15 mg/kg body weight/day
Medium Dose: 60 mg/kg body weight/day
High Dose: 180 mg/kg body weight/day

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 the vehicle
using the same dose volume.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight.
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 copulation. 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:
For determination of the nominal concentration of test item in dosing formulations, samples were retained from all groups in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) (total 16 samples).

In the first week and fifth week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, mid and low dose formulations (total 18 samples)

All samples were analysed on the day of sampling within 6 hours after preparation. Sample quantity for all samples was 35 mL in a 50 mL falcon tube.

All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling

Formulation analysis was performed in accordance with GLP and the procedures followed for the determination of test item concentration in the dosing formulations and control formulation is described in a separate study phase plan issued by the Principal Investigator.
Duration of treatment / exposure:
The female animals were treated with the test item formulation or vehicle on 7 days per week basis for approximately 54 days, i.e. during 14 days of
pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
7 days/week
Details on study schedule:
Arrival of the Test Item: 17 February 2012
Date of Draft Study Plan: 16 July 2012
Date of Final Study Plan: 19 July 2012
Date of 1st amendment 13 August 2012
to study plan
Date of Start of Experiment 02 August 2012
Date of End of Experiment: 01 October 2012
Date of Start of Delegated
Phase (Histopathology): 15 November 2012
Date of End of Delegated
Phase (Histopathology): 05 December 2012

Date of Start of Delegated
Phase (Formulation Analysis) 07 August 2012

Date of End of Delegated
Phase (Formulation Analysis) 20 September 2012

Date of Draft Phase Report
(Histopathology): 07 December 2012

Date of Final Phase Report
(Histopathology): 29 April 2013

Date of Draft Phase Report
(Formulation Analysis): 06 February 2013

Date of Final Phase Report
(Formulation Analysis): 02 April 2013

Date of Draft Report (BSL): 15 February 2013
Date of Final Report (BSL): 02 May 2013

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 60, 180 mg/kg Body weight/day
Basis:
other: Nominal in Sesame oil
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Time schedule: daily
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of
anticipated effects 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.

BODY WEIGHT:
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly
during the treatment period 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)
as well as day 4 post-partum along with pups.

FOOD CONSUMPTION:
Food consumption was measured weekly 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.



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 parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
SACRIFICE
All surviving males were sacrificed on day 29 (minimum total dosing of 28 days). Females were sacrificed on
respective post-partum day 4 along with the pups by using an anaesthesia (ketamine/xylazin, 2:1)
Females not delivered were sacrificed on day 26 after the sperm-positive vaginal smear as an evidence of mating.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which includes 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), and all organs showing macroscopic lesions of all adult animals were
preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were preserved in modified Davidson’s Solution and then transferred in 10 % neutral buffered formalin.

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


HISTOPATHOLOGY / ORGAN WEIGHTS
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately. Organ weights of animals found dead were not taken.

Additional organs (all gross lesions, lung, brain, urinary bladder, stomach, lymph nodes (mesenteric and axillary), small and large intestines
(including Peyer´s patches), trachea, liver, kidneys, thymus, adrenal glands, spleen, heart)of animals which died during the course of the study
were preserved and examined histopathologically in order to determine the cause of death

A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the study which were sacrificed at the end of the treatment period.

A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.

Gross lesions macroscopically identified were examined microscopically.

The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Postmortem examinations (offspring):
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Statistics:
A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item related clinical signs were observed in male and female animals of the MD and HD groups. Predominant clinical signs observed in male and female animals were moving the bedding, piloerection, salivation and abnormal breathing
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item related effect on body weight and food consumption was observed in MD and HD group male and females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item related effect on body weight and food consumption was observed in MD and HD group male and females
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related lesions noted in decedents and the obvious general toxicity in MD and HD dose groups, it is considered that changes noted in these dose groups in male and female reproductive organs are most probably secondary to general toxicity
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reduction in copulation index and fertility index in HD group female was observed

Details on results (P0)

Clinical Observations and Mortality:

In MD males 2 mortalities and in HD females 3 mortalities were observed.

Histopathologically, the deaths of four of these decedents (Male- 23, 28, Female-72 and 74) were considered to be caused by a combination of prominent general toxicity due to bad health and gavaging error/regurgitation into the lung. Death of one high dose female (73) was considered to be due to direct toxicity of the test item alone.
Test item related clinical signs were observed in few male and female animals of the MD and HD groups. Predominant clinical signs observed in male and female animals were moving the bedding, red/brown nasal discharge, slight piloerection, moderate piloerection, slight salivation, moderate salivation and abnormal breathing.


Body Weight Development:

In male, statistically significant decrease in group mean body weight was observed on day of terminal sacrifice in LD, day 7 in HD, day 14, 21, 28 and terminal sacrifice in MD and HD group when compared with controls. The group mean body weight change was statistically significantly decreased from premating day 1 to terminal sacrifice day in all treatment groups, premating day 1-7 in HD group, premating day 7-14 in MD and HD group, premating day 14- mating and post mating 7 in MD and mating and post mating day 7-14 in HD group when compared with controls.

In females, statistically significant decrease in group mean body weight was observed on premating day 14 in HD, gestation day 20 in MD and HD, lactation day 0 in MD and HD and lactation day 4 in HD when compared with controls. The group means body weight change was statistically significantly decreased from premating day 7-14 in HD, gestation day 14-20 and 0-20 in MD and HD group when compared with controls.

This significant effect on male and female body weight development was considered to be treatment related and toxicologically relevant.

Food Consumption:

In males, statistical analysis of food consumption data revealed significant decrease during preamting day 1-7 and 7-14 in MD and HD group when
compared with controls.

In female, statistically significant decrease in group mean food consumption was observed during premating day 1-7 in MD and HD, 7-14 in HD, gestation day 0-7 in MD and HD, gestation day 7-14 in MD, gestation day 14-20 in MD and HD and lactation day 0-4 in HD group when compared with
controls.

This significant effect on male and female food consumption was correlated with decrease in body weight and therefore considered to be treatment
related.

Pathology:

At necropsy, macroscopic examination of the animals revealed few necropsy findings. Findings observed in epididymides were spontaneous as proved by histopathological examination. However, necropsy changes observed in intestine, stomach and other organs of dead animals were considered to be test item related and or to be secondary to the bad terminal health condition and agony of the animals.

Organ Weight:

In males, statistically significant decrease in absolute and relative prostate (with seminal vesicles and coagulating glands) weight was observed in HD group when compared with control. This effect on prostate weight could be considered as test item related.

There was also statistically significant increase in relative testes weight in MD , HD group and relative epididymides weight in HD group was observed when compared with controls. This increase in relative testes and epididymides weight was attributed to the decrease in terminal body weight in MD and HD group animals. Since no effect on absolute testes and epidiymides weight was observed in MD and HD dose group and histopathologically no major findings were observed except in very few males in MD (2) and HD (3), minimal bilateral intraluminal cellular debris in epididymides together with minor degeneration of testicular seminiferous epithelium in one animal each in MD and HD group. These very minor histopathological findings noted in male reproductive organs in this study were not considered to be direct toxic effects due to test item administration, but rather to represent changes secondary to a general toxicity. Therefore, this effect on relative weights was considered to be non adverse and considered not have any impact on organ function.

In females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology:

Two males treated at 60 mg/kg/day and three females treated at 180 mg/kg/day were found dead during the treatment period. The deaths of four of these decedents were considered to be caused by a combination of prominent general toxicity and gavaging error/regurgitation into the lung. Death of one high dose female was considered to be due to direct toxicity of the test item alone.

Besides histopathological changes noted in small intestinal villi, mesenteric lymph nodes and liver, which corresponded to those seen in a 28-day toxicity study with the same test item and were considered as primary toxic lesions, decedents showed also one or more of the following: cortical hypertrophy of the adrenal gland, atrophy/degeneration of lymphoid organs, degeneration of the testicular seminiferous epithelium and atrophy/inactive state of the uterus, all of which were thought to be secondary to the bad terminal health condition and agony of the animals. In two decedents treated at 180 mg/kg/day, histopathological changes were also noted in the nonglandular part of the stomach and indicated local irritation of the test item formulation in the stomach.

At terminal sacrifice, a minimally decreased secretory content of the prostate gland was observed in a dose-related manner at 60 and 180 mg/kg/day. In a small proportion of males treated at 60 and 180 mg/kg/day, minimal bilateral intraluminal cellular debris was noted in the epididymis, in single animals together with minor degeneration of the testicular seminiferous epithelium. In the females, the number of large ovarian corpora lutea and the incidence of regressing placentae in the uterus appeared to be lower at 180 mg/kg/day. Out of the four high dose females noted not to have been pregnant at necropsy, two showed physiological sexual cycling without indication of recent pregnancy, and in the other two physiological sexual cycling was not observed, the uterus being atrophic/inactive.

In view of the test item-related lesions noted in decedents and the obvious general toxicity (lower terminal body weight) in these dose groups, it is considered that changes noted in male and female reproductive organs at 60 and 180 mg/kg/day are most probably secondary to general toxicity rather than being directly caused by the test item.

As a conclusion, under the conditions of this study with limited histopathological evaluation, the NOAEL (No Observed Adverse Effect Level) of pathology was considered to be 15 mg/kg/day.




Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistical analysis of litter weight data revealed significant decrease in group mean litter weight on PND 4 in HD, total litter weight on PND 0 and 4 in MD and HD, male litter weight on PND 4 in HD, female litter weight on PND 4 in MD and HD group
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Litter Data:

Statistical analysis of litter data revealed significant decrease in total number of pups born, total number of live pups on PND 0 and total number of female pups and live pups on PND 4 in HD group when compared with controls. No treatment related effect was observed on number of males, number of females, sex ratio, still birth and runt on PND 0 and number of males and sex ratio on PND 4.

This effect on total number of pups born, total number of live pups on PND 0 and total number of female pups and live pups on PND 4 in HD group could be attributed to the bad general health of the animals from HD group and considered to be secondary to general toxicity rather than being directly caused by the test item.


Litter Weight Data:

Statistical analysis of litter weight data revealed significant decrease in group mean litter weight on PND 4 in HD, total litter weight on PND 0 and 4 in MD and HD, male litter weight on PND 4 in HD, female litter weight on PND 4 in MD and HD group when compared with controls. This effect on litter weight data was considered to be test item related.

Precoital Interval and Duration of Gestation
Statistically significant increase in precoital interval in MD group and increase in duration of gestation in LD group was observed when compared with controls.
Due to lack of dose dependency, this significant effect on precoital interval and duration of gestation was considered to be toxicologically irrelevant. All pregnancies resulted in normal births.


Pre- and Post-Natal Data:

Statistically significant decrease in pre and post natal parameters like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0 was observed in HD group when compared with controls. There was also increase in percent post implantation loss observed in HD group although statistical significance was not achieved. However, percent preimplantation loss in treatment group was comparable with the controls.

This significant effect on HD group pre and post natal parameters could be attributed to the bad general health of the animals from HD group and considered to be secondary to general toxicity rather than being directly caused by the test item.


Reproductive Indices:

No treatment related effect on delivery index and viability index was observed when compared with controls.

Reduced copulation index was observed in Control, LD and HD group. Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C and HD (80 and 40 % respectively) dose group as compared to LD and MD groups (100 %).

Reduction in copulation index and fertility index in HD group was considered to be treatment related effect.


Pup Survival Data:

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study. However, 1 pups from MD group female 62 (pup No. 1) went missing on PND 1 and was presumed to be cannibalized by the dam.

All pups from one HD female (72) were sacrificed on PND 1 due to death of the dam


Pup External Findings:

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed which were not considered to be test item related.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The available study on C16-22-(even numbered)alkylamines (CAS no 68037-92-3) is an OECD 421 reproductive screening test, in which administration of C16-22-(even numbered)alkylamines (CAS no 68037-92-3) to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 15, 60 and 180 mg/kg body weight/day revealed mortalities and findings of toxicological relevance especially in HD group.

Although toxicological findings were found in the HD group, it did not show any indications of reproduction/developmental effects which was not related to maternal toxicity.

Based on the data generated from this reproduction/ developmental toxicity screening test with C16-22-(even numbered)alkylamines, the no observed adverse effect level (NOAEL) for parental animals was considered to be 15 mg/kg body weight/day and NOAEL for developmental toxicity of pup was believed to be 60 mg/kg body weight/day.
Executive summary:

The aim of this study was to assess the possible effect of C16-22-(even numbered)alkylamines on male, female fertility and embryofetal developmentin Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

 In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with15, 60 and 180 mg/kg body weight per dayofC16-22-(even numbered)alkylamines at dose volume of 4 mL/kg body weight. The test item was formulated in sesame oil. Control animals were handled identically as treated groups and received sesame oil in similar volume as treated groups.

The following doses were evaluated:

Control:                              0              mg/kg body weight/day

Low Dose:                         15             mg/kg body weight/day

Medium Dose:                  60            mg/kg body weight/day

High Dose:                        180           mg/kg body weight/day             

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period and post mating period in males where food consumption was not measured.

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for total period of 28 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

After 14 days of premating treatment to both male and female, animals were paired (1:1) for 14 days. The subsequent morning onwards, the vaginal smears of females were checked to confirm the evidence of mating in the form of sperm positive vaginal smears. 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 were weighed within 24 hours of parturition and on day 4 post-partum.

Males and females were sacrificed on treatment day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females (45, 50, 75 and 78) were sacrificed on respective day 26 after the evidence of mating. Females (76 and 77) from HD group were not detected sperm positive during 14 day mating period and sacrificed on day 26 after the completion of mating period. However, females from control (48) and LD (56) although not detected sperm positive during 14 day mating period, animals were pregnant and littered normally.

The wet weight of male and female reproductive organs was taken and preserved in 10 % neutral buffered formalin excepttestes and epididymides which were initially fixed in Modified Davidson’s fixativefor approximately 24 hours before they were transferred to 10% neutral buffered formalin. Histopathological evaluation of the collected tissues was performed in all animals. Organs showing gross alterations were also examined histopathologically.

Summary Results

Clinical Signs and Mortality

            In MD males 2 mortalities and in HD females 3 mortalities were observed.

Histopathologically, the deaths of four of these decedents (Male- 23, 28, Female-72 and 74) were considered to be caused by a combination of prominent general toxicity due to bad health and gavaging error/regurgitation into the lung. Death of one high dose female (73) was considered to be due to direct toxicity of the test item alone.

Test item related clinical signs were observed in few male and female animals of the MD and HD groups. Predominant clinical signs observed in male and female animals were moving the bedding, red/brown nasal discharge, slight piloerection, moderate piloerection, slight salivation, moderate salivation and abnormal breathing.

Body Weight Development

In male, statistically significant decrease in group mean body weight was observed on day of terminal sacrifice in LD, day 7 in HD, day 14, 21, 28 and terminal sacrifice in MD and HD group when compared with controls. The group mean body weight change was statistically significantly decreased from premating day 1 to terminal sacrifice day in all treatment groups, premating day 1-7 in HD group, premating day 7-14 in MD and HD group, premating day 14- mating and post mating 7 in MD and mating and post mating day 7-14 in HD group when compared with controls.

 

In females, statistically significant decrease in group mean body weight was observed on premating day 14 in HD, gestation day 20 in MD and HD, lactation day 0 in MD and HD and lactation day 4 in HD when compared with controls. The group means body weight change was statistically significantly decreased from premating day 7-14 in HD, gestation day 14-20 and 0-20 in MD and HD group when compared with controls.

 

            This significant effect on male and female body weight development was considered to be treatment related and toxicologically relevant.

 

Food Consumption

 In males, statistical analysis of food consumption data revealed significant decrease during preamting day 1-7 and 7-14 in MD and HD group when compared with controls.

 

 In female,statistically significant decrease in group mean food consumption was observed during premating day 1-7 in MD and HD, 7-14 in HD, gestation day 0-7 in MD and HD, gestation day 7-14 in MD, gestation day 14-20 in MD and HD and lactation day 0-4 in HD group when compared with controls.

 

This significant effect on male and female food consumption was correlated with decrease in body weight and therefore considered to be treatment related.

 Litter Weight Data

Statistical analysis of litter weight data revealed significant decrease in group mean litter weight on PND 4 in HD, total litter weight on PND 0 and 4 in MD and HD, male litter weight on PND 4 in HD, female litter weight on PND 4 in MD and HD group when compared with controls. This effect on litter weight data was considered to be test item related.

 Precoital Interval and Duration of Gestation

Statistically significant increase in precoital interval in MD group and increase in duration of gestation in LD group was observed when compared with controls.

 

Due to lack of dose dependency, this significant effect on precoital interval and duration of gestation was considered to be toxicologically irrelevant.All pregnancies resulted in normal births.

            Pre and Post Natal Data

Statistically significant decrease in pre and post natal parameters like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0 was observed in HD group when compared with controls. There was also increase in percent post implantation loss observed in HD group although statistical significance was not achieved. However, percent preimplantation loss in treatment group was comparable with the controls.

 

This significant effect on HD group pre and post natal parameters could be attributed to the bad general health of the animals from HD groupand considered to be secondary to general toxicity rather than being directly caused by the test item. 

  Litter Data

Statistical analysis of litter data revealed significant decrease in total number of pups born, total number of live pups on PND 0 and total number of female pups and live pups on PND 4 in HD group when compared with controls. No treatment related effect was observed on number of males, number of females, sex ratio, still birth and runt on PND 0 and number of males and sex ratio on PND 4.

 

This effect on total number of pups born, total number of live pups on PND 0 and total number of female pups and live pups on PND 4 in HD group could be attributed to the bad general health of the animals from HD groupand considered to be secondary to general toxicity rather than being directly caused by the test item.  

 Reproductive Indices

No treatment related effect on delivery index and viability index was observed when compared with controls.

Reduced copulation index was observed in Control, LD and HD group. Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C and HD (80 and 40 % respectively) dose group as compared to LD and MD groups (100 %).

Reduction in copulation index and fertility index in HD group was considered to be treatment related effect.

Pup Survival Data

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study. However, 1 pups from MD group female 62 (pup No. 1) went missing on PND 1 and was presumed to be cannibalized by the dam.

 

            All pups from one HD female (72) were sacrificed on PND 1 due to death of the dam.

 

Pup External Findings

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed which were not considered to be test item related.

Gross Pathology

 

At necropsy, macroscopic examination of the animals revealed few necropsy findings. Findings observed in epididymides were spontaneous as proved by histopathological examination.However, necropsy changes observed in intestine, stomach and other organs of dead animals were considered to be test item related and or to be secondary to the bad terminal health condition and agony of the animals.

Organ Weight

In males, statistically significant decrease in absolute and relative prostate (with seminal vesicles and coagulating glands) weight was observed in HD group when compared with control. This effect on prostate weight could be considered as test item related.

 

There was also statistically significant increase in relative testes weight in MD , HD group  and relative epididymides weight in HD group was observed when compared with controls. This increase in relative testes and epididymides weight was attributed to the decrease in terminal body weight in MD and HD group animals.  Since no effect on absolute testes and epidiymides weight was observed in MD and HD dose group and histopathologically no major findings were observed except in very few males in MD (2) and HD (3),  minimal bilateral intraluminal cellular debris in epididymides together with minor degeneration of testicular seminiferous epithelium in one animal each in MD and HD group.  These very minor histopathological findings noted in male reproductive organs in this study were not considered to be direct toxic effects due to test item administration, but rather to represent changes secondary to a general toxicity. Therefore, this effect on relative weights was considered to be non adverse and considered not have any impact on organ function.

 

In females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

 

Histopathology

Test item-related lesions noted in decedents and the obvious general toxicity (lower terminal body weight) in 60 and 180 mg/kg/day dose groups, it is considered that changes noted in these dose groups in male and female reproductive organs are most probably secondary to general toxicity rather than being directly caused by the test item.

 

As a conclusion, under the conditions of this study with limited histopathological evaluation, the NOAEL (No Observed Adverse Effect Level) of pathology was considered to be 15 mg/kg/day.

 

  Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD group were 105.0%, 105.7% and 107.5% of the nominal concentration, respectively.

 

Homogeneity of formulation samples was determined in study weeks 1 and 5 for all dose groups. The mean recoveries observed for LD group were 110.7% and 105.3%, for MD group 105.8% and 104.7%, and for HD group 102.5% and 104.5% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) in LD group were 3.3% and 1.5%, in MD group 1.5% and 0.6% and in HD group 4.1% and 1.0%.

 

  Conclusions

In conclusion, the repeated dose administration of the test itemto the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 15, 60 and 180 mg/kg body weight/dayrevealedmortalities and findings of toxicological relevance especially in HD group.

Based on the data generated from this reproduction/ developmental toxicity screening test withC16-22-(even numbered)alkylamines, the no observed adverse effect level (NOAEL) for parental animals was considered to be 15 mg/kg body weight/day and NOAEL for developmental toxicity of pup was believed to be 60 mg/kg body weight/day.