N,N,N',N'-tetramethylhexamethylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetramethylhexamethylenediamine

EC Number:

203-842-9

EC Name:

N,N,N',N'-tetramethylhexamethylenediamine

Cas Number:

111-18-2

Molecular formula:

C10H24N2

IUPAC Name:

[6-(dimethylamino)hexyl]dimethylamine

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N´,N´-Tetramethyl-1,6-hexanediamine
- Physical state: Colourless to yellow clear liquid on arival at testing facility and colourless liquid at first use.
- Analytical purity: 100 %
- Lot/batch No.: 000STD77L0
- Expiration date of the lot/batch: 05 August 2013
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability was found to be 24 hours at room temperature in the concentration range of 0.75 to 7.5 mg/mL.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 - 212 g for males and 156 - 164 g 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral 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.

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

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

Examinations

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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.

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.

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Samples of all the tissues (all parental animals) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

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.

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.

Results and discussion

Results of examinations

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
At the daily clinical examination the following observations were recorded:
- Treated males did not show any relevant clinical signs throughout the study.
- Before the mating period one female (animal no.: 88510071) receiving 75 mg/kg bw/day showed rales for 2 days. This clinical sign was not considered to be compound-related due to its transient appearance.
- 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 no. 88510031 receiving 7.5 mg/kg bw/day.
- One female (animal no.: 88510075) receiving 75 mg/kg bw/day showed hunched posture, piloerection and pale aspect during the post partum period.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Means of body weight and body weight gain were comparable between control and treated groups both in males and females throughout the study.
Decreases in body weight and in body weight gain noted during the post partum phase in control and treated females were considered as a normal consequence of the parturition occurred in the females. Female no. 88510075 showed markedly decreased body weight gain toward the end of the study, leading to a terminal body weight of approximately 187 g versus 255 g mean weight in control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The most relevant finding observed was a marked and statistically significantly decrease of eosinophils in animals of both sexes receiving 75 mg/kg bw/day. In four out of five females no eosinophils were detected.
Animal no. 88510075 showed severe anaemia, leucocytosis and thrombocytopenia.
In all other cases, where statistically significant differences occurred (lower Mean corpuscular Hb concentration (MCHC) values and higher reticulocyte values in males at 75 mg/kg bw/day; lower lymphocyte and higher granulocyte values (%) in males at 7.5 mg/kg bw/day), they were minor, occurred without dose-dependency and/or were observed in one sex only. Therefore, these differences were considered not to be compound related.
Animal no. 88510076 showed slightly higher erythrocyte, haemoglobin, and haematocrit values. Since only one animal was affected, these findings were not considered to be treatment-related.
Coagulation test: No changes were recorded.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Phosphorus concentrations were statistically significantly higher in males at 75 mg/kg bw/day. However, in absence of these effects in females and any other changes in males, this difference was not considered as adverse.
On the individual animal level, female no. 88510075 had lower protein concentrations, higher triglyceride and slightly higher glucose levels.
The statistically significantly lower globulin values in females dosed with 7.5 mg/kg bw/day and the lower sodium levels in males treated with 75 mg/kg bw/day were of low magnitude and/or not dose-related, and therefore not considered as compound-related.
The same applies for the minor differences observed in two males and two females at 75 mg/kg bw/day when compared to controls:
- male no. 88510076: slightly higher alanine aminotransferase, aspartate aminotransferase, phosphorus, bile acids, calcium and potassium;
- male no. 88510068: slightly higher values of glucose, phosphorus, calcium and potassium;
- female no. 88510071: slightly higher aspartate aminotransferase, phosphorus and potassium;
- female no. 88510067: slightly higher potassium values.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
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. Statistically significantly lower grip strength was recorded in all treated females when compared to thecontrol group. These variations were considered of no toxicological relevance since they were not dose-related and observed in only one sex. Moreover, the control female group showed an unexpected mean high value, in particular in the first trial.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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.
Among other findings, female no. 88510075 showed thickened glandular and non glandular stomach, dark red areas and dark contents at the stomach.
The remaining macroscopic changes were considered incidental and not treatment-related.

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 female no 88510075. 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 our experimental conditions.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

22.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

2

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed at the highest dose level of 75 mg/kg bw/d

3

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 effects were observed at the highest dose level of 75 mg/kg bw/d

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Haematology Parameters - Group mean data – Males:

Parameters (units)		Group
		1	2	3	4
Red blood cell count (10^6/ul)	Mean SD n	8.416 0.462 5	8.302 0.326 5	8.522 0.330 5	9.028 0.871 5
HAEMOGLOBIN (g/dl)	Mean SD n	14.86 0.45 5	14.62 0.56 5	15.16 0.39 5	15.24 1.58 5
Haematocrit (%)	Mean SD n	44.22 0.91 5	43.72 1.70 5	45.80 1.67 5	48.04 7.07 5
Mean Red Blood Cell Volume (fl)	Mean SD n	52.64 2.38 5	52.66 1.62 5	53.74 0.79 5	53.04 3.55 5
Mean Corpuscular HB (pg)	Mean SD n	17.70 1.23 5	17.62 0.86 5	17.80 0.42 5	16.90 0.99 5
Mean Corpuscular HB (g/dl)	Mean SD n	33.58 0.90 5	33.46 0.88 5	33.12 0.63 5	31.88 1.44 5
Reticulocytes %	Mean SD n	1.668 0.192 5	2.096 0.243 5	1.954 0.214 5	2.186 0.418 5
Reticulocytes (10^9/L)	Mean SD n	139.90 12.59 5	173.78 18.48 5	166.80 20.15 5	197.86 45.97 5
White blood cell count (10^3/ul)	Mean SD n	5.854 1.044 5	4.354 0.791 5	4.912 1.188 5	6.568 1.110 5
Neutrophils (10^3/ul)	Mean SD n	0.852 0.202 5	1.070 0.304 5	1.058 0.332 5	1.174 0.323 5
Lymphocytes (10^3/ul)	Mean SD n	4.700 0.956 5	3.004 0.598 5	3.606 1.083 5	5.120 1.039 5
Monocytes (10^3/ul)	Mean SD n	0.154 0.033 5	0.160 0.041 5	0.110 0.049 5	0.154 0.069 5
Eosinophils (10^3/ul)	Mean SD n	0.064 0.015 5	0.060 0.035 5	0.048 0.015 5	0.018 0.008 5
Basophils (10^3/ul)	Mean SD n	0.034 0.017 5	0.024 0.009 5	0.028 0.015 5	0.050 0.026 5
Large unstained cells (10^3/ul)	Mean SD n	0.050 0.042 5	0.040 0.021 5	0.066 0.038 5	0.054 0.032 5
Neutrophils (%)	Mean SD n	14.76 3.38 5	24.70 5.94 5	22.46 7.51 5	18.22 5.64 5
Lymphocytes (%)	Mean SD n	80.00 4.06 5	68.92 5.50 5	72.50 8.02 5	77.66 4.85 5
Monoocytes (%)	Mean SD n	2.38 0.41 5	3.68 0.58 5	2.22 0.81 5	2.24 0.63 5
Eosinophils (%)	Mean SD n	1.16 0.44 5	1.28 0.58 5	0.96 0.18 5	0.30 0.12 5
Basophils (%)	Mean SD n	0.58 0.18 5	0.54 0.11 5	0.60 0.21 5	0.74 0.21 5
Large unstained cells (%)	Mean SD n	0.82 0.60 5	0.88 0.46 5	1.30 0.58 5	0.84 0.43 5
Platelets (10^3/ul)	Mean SD n	788.0 79.4 5	729.6 59.7 5	733.6 46.2 5	827.4 91.7 5

 

Haematology Parameters - Group mean data – Females:

Parameters (units)		Group
		1	2	3	4
Red blood cell count (10^6/ul)	Mean SD n	6.734 0.336 5	6.750 0.186 5	7.24 0.199 4	6.000 2.445 5
HAEMOGLOBIN (g/dl)	Mean SD n	12.98 0.40 5	12.78 0.64 5	13.65 0.31 4	11.54 4.77 5
Haematocrit (%)	Mean SD n	38.62 1.43 5	37.98 1.19 5	40.40 1.02 4	34.52 13.44 5
Mean Red Blood Cell Volume (fl)	Mean SD n	57.40 1.91 5	56.26 1.16 5	55.78 1.09 4	58.52 3.24 5
Mean Corpuscular HB (pg)	Mean SD n	19.32 0.46 5	18.96 0.67 5	18.88 0.38 4	19.12 0.74 5
Mean Corpuscular HB (g/dl)	Mean SD n	33.68 0.70 5	33.68 0.64 5	33.85 0.17 4	32.78 2.38 5
Reticulocytes %	Mean SD n	5.884 1.549 5	6.826 1.206 5	4.700 0.827 4	8.900 8.297 5
Reticulocytes (10^9/L)	Mean SD n	392.34 84.41 5	459.42 73.50 5	340.18 60.82 4	372.50 48.17 5
White blood cell count (10^3/ul)	Mean SD n	3.214 1.173 5	3.166 0.665 5	3.005 0.568 4	1.680 1.625 5
Neutrophils (10^3/ul)	Mean SD n	1.008 0.377 5	1.102 0.399 5	0.830 0.198 4	1.630 1.625 5
Lymphocytes (10^3/ul)	Mean SD n	2.110 1.193 5	1.960 0.286 5	2.078 0.425 4	2.830 1.643 5
Monocytes (10^3/ul)	Mean SD n	0.048 0.020 5	0.050 0.021 5	0.050 0.008 4	0.090 0.107 5
Eosinophils (10^3/ul)	Mean SD n	0.020 0.012 5	0.022 0.011 5	0.018 0.010 4	0.002 0.004 5
Basophils (10^3/ul)	Mean SD n	0.010 0.010 5	0.008 0.004 5	0.015 0.006 4	0.014 0.005 5
Large unstained cells (10^3/ul)	Mean SD n	0.022 0.019 5	0.024 0.015 5	0.020 0.008 4	0.020 0.014 5
Neutrophils (%)	Mean SD n	37.28 22.63 5	34.08 5.44 5	27.73 4.19 4	32.04 6.56 5
Lymphocytes (%)	Mean SD n	59.66 22.48 5	62.74 5.84 5	68.95 4.28 4	65.40 6.80 5
Monoocytes (%)	Mean SD n	1.52 0.51 5	1.54 0.62 5	1.65 0.31 4	1.58 0.59 5
Eosinophils (%)	Mean SD n	0.62 0.26 5	0.64 0.15 5	0.55 0.21 4	0.06 0.09 5
Basophils (%)	Mean SD n	0.30 0.21 5	0.24 0.05 5	0.53 0.33 4	0.40 0.19 5
Large unstained cells (%)	Mean SD n	0.60 0.34 5	0.72 0.36 5	0.58 0.21 4	0.48 0.25 5
Platelets (10^3/ul)	Mean SD n	1415.2 339.4 5	1345.4 168.3 5	1088.5 138.6 4	1311.4 380.7 5

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study and based on the results obtained, the NOAEL for N,N,N´,N´-Tetramethyl- 1,6-hexanediamine was considered to be 22.5 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.