Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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:
April/May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern, guideline and GLP-compliant study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 - 10 weeks old.
- Weight at study initiation: 195 - 212g for males and 156 - 164g for females.
- Fasting period before study: Groups were food deprived prior to the start of testing.
- Housing: From arrival to pairing animals were housed up to 5/sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor.
During mating animals were housed one male to one female in clear polycarbonate cages measuring 42.5x26.6x18 cm with a stainless steel mesh lid and floor.
After mating the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages measuring 42.5x26.6x18 cm for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet was offered ad libitum throughout the study, except to those groups which were food-deprived during testing.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light (artificial light).

IN-LIFE DATES: From 05 March 2012: To: 28 April 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 - 10 weeks old.
- Weight at study initiation: 195 - 212g for males and 156 - 164g for females.
- Fasting period before study: Groups were food deprived prior to the start of testing.
- Housing: From arrival to pairing animals were housed up to 5/sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor.
During mating animals were housed one male to one female in clear polycarbonate cages measuring 42.5x26.6x18 cm with a stainless steel mesh lid and floor.
After mating the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages measuring 42.5x26.6x18 cm for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet was offered ad libitum throughout the study, except to those groups which were food-deprived during testing.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light (artificial light).

IN-LIFE DATES: From 05 March 2012: To: 28 April 2012
Details on mating procedure:
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray). Each cage tray was checked each morning for the presence of copulation plugs. The female was paired with the same male until positive identification of copulation occurs or 14 days had elapsed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable. Results of the analyses were within the limits of acceptance. The stability was found to be 24 hours at room temperature in the concentration range of 0.75 to 7.5 mg/mL.
Duration of treatment / exposure:
Male test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and up to the day before necropsy.
Female test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Frequency of treatment:
Daily during the exposure period
Details on study schedule:
Male test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and up to the day before necropsy.
Female test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Remarks:
Doses / Concentrations:
0, 7.5, 22.5, 75 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Positive control:
Not applicable; not required for this type of study
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena for a minimum of 3 minutes. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the necropsy procedure.
- Anaesthetic used for blood collection: Yes- isofluorane anaesthesia
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked include: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differetntial leucocyte count (including neutrophils, lymphocytes, eosinophils, basophils, monocytes and large unstained cells) and platelets.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During the necropsy procedure.
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked include: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, phosphorous, total biliubin, total cholesterol, bile acids, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium and chloride.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the study, towards the end of treatment.
- Dose groups that were examined: males and 5 females were randomly selected from each group for examination.
- Battery of functions tested: sensory activity, grip strength and motor activity.

OTHER:
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made to determine any anomalies of the oestruss cycle and the pre-coital interval.

Parturition check and duration of gestation: A parturition check was performed from Day 20 to Day 25 post coitum. Gestation periods were taken as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Hormone determinations: Approximately 1.2 mL of blood samples were drawn under isofluorane anaesthesia from the same animals in the food-deprived group. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature.
The serum obtained was divided in several aliquots and frozen at -80°C.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine anomalies of the oestrous cycle.
Sperm parameters (parental animals):
A detailed qualitative examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952. The PAS-stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, not relevant

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death wa determined for pups born or found dead.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes. All females were examined for external and internal abnormalities, the number of visible implantation sites (pregnant animals) and the number of corpura lutea (pregnant animals). The uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY: Yes. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.

The histopathological examination was restricted to the cervix, clitoral gland, ovaries, uterus and vagina from all parental females, the coagulating gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes from all parental males, tissues selected from 5 males and 5 females selected at random in the control and high dose groups killed at term and all abnomalities in all groups.
Postmortem examinations (offspring):
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if ‘n’ was more than 5.
Reproductive indices:
Copulatory Index (M/F)
Fertility Index (M/F)
Offspring viability indices:
Not calculated
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs including hunched posture, piloerection and pale aspect were observed in one female at 75 mg/kg bw/d
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
One female at 75 mg/kg bw/d showed lower body weight gain towards the end of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
One female at 75 mg/kg bw/d showed lower body weight gain towards the end of the study.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One female at 75 mg/kg bw/d showed thickened glandular and non glandular stomach, dark red areas and dark contents at the stomach.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mucosal ulceration, oedema, chronic inflammation in the stomach and moderate atrophy in the thymus were observed in one high dose level female
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
No mortality occurred throughout the study. At the daily clinical examination treated males did not show any relevant clinical signs throughout the study.
Prior to the mating period one female receiving the highest dose level (75 mg/kg/day) showed rales for 2 days.
No relevant clinical signs were noted in control and treated females during the post coitum period.
Red staining on the cage tray was observed on Day 23 post coitum in female one female receiving 7.5 mg/kg/day.
One female receiving 75 mg/kg/day showed hunched posture, piloerection and pale aspect during the post partum period.

BODY WEIGHT AND WEIGHT GAIN

Means of body weight and body weight gain were comparable between control and treated groups both in males and females throughout the study. Reduced weight gain was seen in one high dose female.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

Food consumption was unaffected by treatment in both sexes during the study.

HAEMATOLOGY

A marked decrease of eosinophils was seen in animals of both sexes receiving 75 mg/kg/day. In 4/5 of the females no eosinophils were detected.


CLINICAL CHEMISTRY

There were no effects of treatment.

NEUROBEHAVIOUR
Motor activity recorded at the end of treatment was comparable between control and treated groups. Variations recorded in the sensory reaction to stimuli measurements, performed at the end of the treatment period, were considered of no toxicological significance.

ORGAN WEIGHTS
A decrease in absolute brain weight in males receiving 7.5 mg/kg/day was observed. In females receiving 22.5 mg/kg/day or more, there was a dose-related decrease in absolute thyroid weight. There was a decrease in relative spleen weight in males receiving 75 mg/kg/day. Females treated with 22.5 mg/kg/day showed a decrease in relative thyroid weight.

GROSS PATHOLOGY

Reduced size of prostate glands was observed in three males of each treated group associated with reduction of seminal vesicles and testis (left) in one low dose male. Since there was no dose-dependency and in the absence of histopathological correlates, these findings were considered not to be treatment-related. One female at 75 mg/kg bw/d showed thickened glandular and non glandular stomach, dark red areas and dark contents at the stomach.

HISTOPATHOLOGY

Mucosal ulceration, oedema, chronic inflammation in the stomach and moderate atrophy in the thymus were observed in one high dose level female; the latter change could be considered to be stress related. No further changes were seen in selected organs/tissues evaluated in males or females
receiving N,N,N´,N´-Tetramethyl-1,6-hexanediamine which could be related to treatment. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.

A detailed qualitative examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
The PAS-stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
22.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects were seen at the highest dose level
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
No differences in total and live litter size, mean pup weight and sex ratio were noted between groups at birth and on Day 4 post partum. Mean litter weight of females receiving 75 mg/kg bw/day was lower than control group on both Day 1 post partum (-17%) and Day 4 post partum (-20%). The change reached statistical significance on Day 4 post partum. This difference was due to two reasons: Firstly, the control live litter size (12.2) was higher than the ascending dose groups (10.8, 11.2, and 10.5); secondly, female no. 88510075 gave birth to pups with a low birth weight, which lost body weight during the postnatal phase. In light of the impaired general condition of the dam, this finding was not considered to represent a compound-related effect on reproduction.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity
Reproductive effects observed:
not specified
Conclusions:
Under the conditions of this study, no direct effect of treatment on reproduction or fertility was seen under the conditions of this study.
Executive summary:

The purpose of this study was to generate information concerning toxic effects on rats of both sexes after repeated dosing with N,N,N´,N´-Tetramethyl-1,6-hexanediamine, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated. Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. At necropsy a detailed external and internal examination was performed. The histopathological examination, including identification of the stages of the spermatogenic cycle (males) was carried out in five males of control and high dose groups, selected randomly. In addition, reproductive organs such as coagulating gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes were examined histopathologically on all parental males. No relevant signs of toxicological significance were observed at the clinical examination including body weights and food consumption. Fertility index and copulatory index were unaffected by treatment. At haematological evaluation the only alteration noted in males receiving 75 mg/kg bw/d was a decrease in eosinophils. No adverse effects were observed at clinical chemistry examination. Reduced size of prostate glands was observed in three males of each treated group, associated with reduction of seminal vesicles and testis (left) in one low dose male. Since there was no dose-dependency and in the absence of histological correlates, these findings were considered not to be treatment-related.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum. During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated. Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. The histopathological examination was carried out in five females of control and high dose groups, selected randomly. In addition, reproductive organs such as cervix, clitoral gland, ovaries, uterus and vagina were examined histopathologically on all parental females. Clinical signs of pups as well as necropsy examination of pups sacrificed at termination or unscheduled deaths were recorded. At necropsy a detailed external and internal examination was performed. Litter data, sex ratios and gestation length were recorded. One female at 75 mg/kg bw/d showed clinical signs and decreased body weight gain towards the end of the study, and did not raise its offspring properly. Haematology parameters revealed anaemia, leucocytosis, and thrombocytopenia. These effects correlate with the histopathological findings of ulceration and chronic inflammation at the stomach. In addition, a moderate atrophy of the thymus was also observed. The latter change could be considered to be stress related. It is likely that the above mentioned findings are the primary cause of the worsening of general condition of this animal towards study end. Although only one female was affected, it cannot fully be excluded that the high pH value of the test compound had contributed to the inflammation process in the stomach, either directly or secondary to a pre-existing lesion. At least, the postnatal effects on the pups were considered to be the consequence of the impaired maternal health status. Consistent with males, haematological examinations revealed decreased eosinophil values at 75 mg/kg bw/d, which may be the consequence of stress at this dose level. No further compound-related effects were noted, including mating, gestation, birth, nursing behaviour, and postnatal development of the offspring.

Based on the results of the study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 22.5 mg/kg bw/d for males and females. The NOAEL for reproductive and developmental toxicity was considered to be 75 mg/kg bw/d for males and females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern screening study (OECD 422) is available for this endpoint.
Additional information
Justification for selection of Effect on fertility via oral route:
Only one study for this endpoint

Effects on developmental toxicity

Description of key information
No evidence of reprodcutive toxicity or specific developmental toxicity was seen in a screening study at the parentally toxic dose level of 75 mg/kg bw/d.  
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March-April 2012
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 9 - 10 weeks old.
- Weight at study initiation: 195 - 212g for males and 156 - 164g for females.
- Fasting period before study: Groups were food deprived prior to the start of testing.
- Housing: From arrival to pairing animals were housed up to 5/sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor.
During mating animals were housed one male to one female in clear polycarbonate cages measuring 42.5x26.6x18 cm with a stainless steel mesh lid and floor.
After mating the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages measuring 42.5x26.6x18 cm for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet was offered ad libitum throughout the study, except to those groups which were food-deprived during testing.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light (artificial light).


IN-LIFE DATES: From 05 March 2012: To: 28 April 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of N,N,N´,N´-Tetramethyl-1,6-hexanediamine was dissolved in the vehicle (purified water) and brought to the final volume appropriate for each concentration (concentrations of 0.75, 2.25 and 7.5 mg/mL) and shaken manually until complete dissolution. The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable. Results of the analyses were within the limits of acceptance. The stability was found to be 24 hours at room temperature in the concentration range of 0.75 to 7.5 mg/mL.
Samples of the formulations prepared on Week 1 and last Week (when all females were present) were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.
Chemical analysis was carried out in the range from 1.0 to 2.5 mg/mL. In the present study the validation of the method was performed for the lower concentration 0.1 mg/mL.
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1 male to 1 female
- Length of cohabitation: The female was paired with the same male until positive identification of copulation occurs or 14 days have elapsed.
- Proof of pregnancy: sperm in vaginal smear
Duration of treatment / exposure:
Male test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and up to the day before necropsy.
Female test animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Frequency of treatment:
Daily during the exposure period
Duration of test:
Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and up to the day before necropsy.
Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Remarks:
Doses / Concentrations:
0, 7.5, 22.5, 75 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena for a minimum of 3 minutes. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed weekly from allocation to pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Females with live pups were sacrificed on Day 4 post partum.
- Organs examined: All females were examined for external and internal abnormalities, number of visible implantation sites (pregnant animals) and the number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.
Reproductive organs including the cervix, clitoral gland, ovaries, uterus and vagina from all parental females were examined.

OTHER:
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made to determine any anomalies of the oestruss cycle and the pre-coital interval.

Parturition check and duration of gestation: A parturition check was performed from Day 20 to Day 25 post coitum. Gestation periods were taken as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if ‘n’ was more than 5.
Indices:
The reproductive indices calculated include pre-coital interval, fertility index in males and the copulatory and fertility index in females. In females, the percentage pre-birth loss and pup loss at birth was also calculated.
Historical control data:
No data.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Reduced eosinophil counts; gastric ulceration and secondary effects (including reduced weight gain) in one female

Details on maternal toxic effects:
Reduced eosinophil counts; gastric ulceration and secondary effects (including reduced weight gain) in one female
Dose descriptor:
NOAEL
Effect level:
22.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified

Corpora lutea, implantations, total litter size and pre-birth loss (percentage) were similar in control and treated groups. No differences in total and live litter size, mean pup weight and sex ratio were noted between groups at birth and on Day 4 post partum. Mean litter weight of females receiving 75 mg/kg bw/day was lower than control group on both Day 1 post partum (-17%) and Day 4 post partum (-20%). The change reached statistical significance on Day 4 post partum. This difference was due to two reasons: Firstly, the control live litter size (12.2) was higher than the ascending dose groups (10.8, 11.2, and 10.5); secondly, female no. 88510075 gave birth to pups with a low birth weight, which lost body weight during the postnatal phase. In light of the impaired general condition of the dam, this finding was not considered to represent a compound-related effect on reproduction.

Conclusions:
No evidence of specific developmental toxicity was seen in this screening study at the parentally toxic dose level of 75 mg/kg bw/d.
Executive summary:

The purpose of this study was to generate information concerning toxic effects on rats of both sexes after repeated dosing with N,N,N´,N´-Tetramethyl-1,6-hexanediamine, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated. Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. At necropsy a detailed external and internal examination was performed. The histopathological examination, including identification of the stages of the spermatogenic cycle (males) was carried out in five males of control and high dose groups, selected randomly. In addition, reproductive organs such as coagulating gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes were examined histopathologically on all parental males. No relevant signs of toxicological significance were observed at the clinical examination including body weights and food consumption. Fertility index and copulatory index were unaffected by treatment. At haematological evaluation the only alteration noted in males receiving 75 mg/kg bw/d was a decrease in eosinophils. No adverse effects were observed at clinical chemistry examination. Reduced size of prostate glands was observed in three males of each treated group, associated with reduction of seminal vesicles and testis (left) in one low dose male. Since there was no dose-dependency and in the absence of histological correlates, these findings were considered not to be treatment-related.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum. During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated. Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. The histopathological examination was carried out in five females of control and high dose groups, selected randomly. In addition, reproductive organs such as cervix, clitoral gland, ovaries, uterus and vagina were examined histopathologically on all parental females. Clinical signs of pups as well as necropsy examination of pups sacrificed at termination or unscheduled deaths were recorded. At necropsy a detailed external and internal examination was performed. Litter data, sex ratios and gestation length were recorded. One female at 75 mg/kg bw/d showed clinical signs and decreased body weight gain towards the end of the study, and did not raise its offspring properly. Haematology parameters revealed anaemia, leucocytosis, and thrombocytopenia. These effects correlate with the histopathological findings of ulceration and chronic inflammation at the stomach. In addition, a moderate atrophy of the thymus was also observed. The latter change could be considered to be stress related. It is likely that the above mentioned findings are the primary cause of the worsening of general condition of this animal towards study end. Although only one female was affected, it cannot fully be excluded that the high pH value of the test compound had contributed to the inflammation process in the stomach, either directly or secondary to a pre-existing lesion. At least, the postnatal effects on the pups were considered to be the consequence of the impaired maternal health status. Consistent with males, haematological examinations revealed decreased eosinophil values at 75 mg/kg bw/d, which may be the consequence of stress at this dose level. No further compound-related effects were noted, including mating, gestation, birth, nursing behaviour, and postnatal development of the offspring.

Based on the results of the study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 22.5 mg/kg bw/d for males and females. The NOAEL for reproductive and developmental toxicity was considered to be 75 mg/kg bw/d for males and females.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern screening study (OECD 422) is available for this endpoint.
Additional information

No evidence of reprodcutive toxicity or specific developmental toxicity was seen in a screening study at the parentally toxic dose level of 75 mg/kg bw/d. One female at 75 mg/kg bw/d showed clinical signs and lower body weight gain towards the end of the study. Offspring of this rat were not reared properly and lost weight from Day 1 to Day 4 post partum. Hematological examinations revealed anemia, leucocytosis and thrombopenia. Mucosal ulceration, oedema and chronic inflammation of the stomach were observed in this animal at histological examination. In addition, moderate atrophy of the thymus was also observed. Although only one animal was affected, it cannot fully be excluded that these findings have been caused primarily by a local corrosive action of the test compound (high pH value), either directly or secondary to a pre-existing lesion.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available for this endpoint.

Justification for classification or non-classification

Findings in the available study do not trigger classification of the substance for reproductive toxicity according to current guidance on CLP classification.