Diallylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (gavage) combined repeat dose toxicity study with reproductive/developmental toxicity screening test in the rat (OECD 422)

The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development).

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 15, 30 and 75 mg/kg bw/day. An additional male was included in the high dose group to replace a male which was terminated on Day 3 because this death was unrelated to treatment. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study (Day 17 for the additional high dose male and respective partner), with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum.

Reproductive Performance:

Mating: No treatment-related effects were detected in mating performance.

Fertility: No treatment-related effects were detected in fertility.

Two females treated with 15 mg/kg bw/day and two females treated with 30 mg/kg bw/day were not pregnant following positive evidence of mating. No histopathological changes were evident in the female or male reproductive organs which could have been the cause of the infertility.

Gestation length:

Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

One control female that had a total litter loss had a gestation length of 24½ days. It is not unusual for females with small litters and/or extended gestation periods to fail to maintain their litter. This was considered to be incidental as it was from a control group female and no similar effects in other groups.

Mating performance and fertility was unaffected by treatment.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 75 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 19 October 2015 and 22 November 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

No deviations where the scientific integrity of the study is affected.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 302 to 370g, the females weighed 185 to 223g
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: The animals were acclimatized for five days during which time their health status was assessed. A total of ninety seven animals (forty nine males and forty eight females) were accepted into the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): target ranges for temperature were 22 ± 3 °C
- Humidity (%): target ranges for t relative humidity were 50 ± 20%
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

The in-life phase of the study was conducted between 01 December 2015 (first day of treatment) and 24 January 2016 (final day of necropsy).

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited. Results show the formulations to be stable for twenty-one days days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulation were taken on three occasions and analyzed for concentration of Diallylamine.

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.

The homogeneity and stability was confirmed for Test Item in Arachis Oil BP formulations at nomincal concentrations of 2.5 mg/ml and 100 mg/ml when stored refrigerated for 21 days.

The mean concentration of Test Item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females.

Frequency of treatment:

The test item was administered daily

Details on study schedule:

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment (excluding the additional high dose male) and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15 (Day 17 for the additional high dose male and respective partner), animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Days 41 (additional high dose group male only), Day 43 or Day 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Low treatment group

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Intermediate treatment group

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

High treatment group

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat.
The oral (gavage) administration of Diallylamine at dose levels of 100, 75 and 30 mg/kg bw/day for a period of fourteen consecutive days, resulted in treatment related effects in animals of either sex from all treatment groups. The body weight effect detected at 100 mg/kg bw/day was sufficient to exclude this level from consideration as the high dose level for any further studies. The effects at 75 and 30 mg/kg bw/day however were insufficient to exclude these dose levels from further investigation.

Positive control:

None

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing and one hour after dosing (except for females during parturition where applicable). Additional observations were also made four hours following dosing (not at weekends or public holidays). All observations were recorded.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination (daily body weights were performed on High dose male animals from Day 9 until Day 19 due to reduced body weight gains and actual body weight losses being noted) and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Gravimetric measurement of water consumption was performed daily during the pre-pairing treatment phase of the study.

SPECIALIST EVALUATION:
FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment (excluding the additional high dose male) and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIORAL ASSESSMENT:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

SENSORY REACTIVITY:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex , Finger approach

REPRODUCTIVE PERFORMANCE:
MATING: See details on mating procedure section.

PREGNANCY AND PARTURITION:
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

IN-LIFE SAMPLING AND ANALYSIS:
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++) Creatinine (Creat)

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination 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. Any cell-or stage-specificity of testicular findings was noted.

Litter observations:

LITTER DATA:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

NECROPSY:
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 41 (additional high dose group male only), Day 43 or 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown with * were weighed from all remaining animals:
Adrenals Pituitary (post-fixation)*
Brain Prostate and Seminal Vesicles*
Epididymides* Spleen
Heart Testes*
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries* Uterus (weighed with Cervix)*

HISTOPATHOLOGY:
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown with * were preserved from all remaining animals:
Adrenals Muscle (skeletal)
Aorta (thoracic) Ovaries*
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary*
Brain (including cerebrum, cerebellum and pons) Prostate*
Caecum Rectum
Coagulating gland* Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles*
Epididymides* Skin (hind limb)
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
Eyes*
Gross lesions* Spleen
Heart Stomach
Ileum (including peyer’s patches) Testes*
Jejunum Thyroid/Parathyroid
Kidneys Trachea
Liver Thymus
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix*
Mammary gland* Vagina*

Postmortem examinations (offspring):

NECROPSY:
Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent.

Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Statistical analysis:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module:

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval:
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices:
For each group the following were calculated:
Mating Index (%) = Number of animals mated / Number of animals paired x 100
Pregnancy Index (%) = Number of pregnant females / Number of animals mated x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100

Offspring viability indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = Number of corpora lutea - Number of implanti on sites / Number of corpora lutea x 100
Post-implantation loss (%) = Number of implanti on sites - Total number of offspring born / Number of implanti on sites x 100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4 / Number of offsrping alive on Day 1 x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Number of male offspring / Total number of offspring x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 75 mg/kg bw/day showed incidences of increased salivation from Day 9 (males) and Day 13 (females) until termination.
No such effects were evident in animals of either sex treated with 30 or 15 mg/kg bw/day.
One control male had noisy respiration between Days 21 and 29. This was considered to be incidental and a result of the method of administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male treated with 75 mg/kg bw/day was killed in extremis on Day 3 due to a physical injury that was not related to treatment. This male was replaced on the study by Male No. 97.
There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 and 30 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in the majority of males treated with 75 mg/kg bw/day and in three males treated with 30 mg/kg bw/day. Recovery was evident in males thereafter and body weight gains in subsequent weeks (excluding Week 5) for treated males were actually higher than control males (statistical significance was achieved during Weeks 2 (all groups), 4 (high dose group) and 6 (intermediate and high dose groups). As a consequence of the initial body weight losses however, overall body weight gain for males treated with 75 mg/kg bw/day was still lower than controls.
No such effects were detected in males treated with 15 mg/kg bw/day.
Females treated with 75 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Actual body weight losses were evident in nine females on Day 8. Recovery was evident during gestation however, a slight reduction in body weight gain was evident in these females during lactation although statistical significance was not achieved.
No such effects were detected in females treated with 30 or 15 mg/kg bw/day.
Females treated with 15 mg/kg bw/day showed a statistically significant increase in body weight gain during lactation. An increase in body weight gain is not considered to reflect an adverse effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males treated with 75 mg/kg bw/day showed a reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. Food conversion efficiency was also reduced in these males during the first week of treatment. A slight reduction in food conversion efficiency was evident in males treated with 30 mg/kg bw/day during the first week of treatment, however this was considered to be the result of lower body weight gain in these males during this week. Recovery was evident thereafter. No such effects were detected in males treated with 15 mg/kg bw/day.
Females treated with 75 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency during both weeks of maturation. Food consumption was also statistically significantly reduced during the first week of gestation in these females. Recovery was evident thereafter. No such effects were detected in females treated with 30 or 15 mg/kg bw/day.

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

effects observed, treatment-related

Description (incidence and severity):

Females treated with 75 and 30 mg/kg bw/day showed an increase in overall water consumption during maturation.
No such effects were detected in females treated with 15 mg/kg bw/day or treated males.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects detected in the hematological parameters examined.
Statistical analysis of the data did not reveal any significant intergroup differences.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males from all treatment groups showed a statistically significant reduction in aspartate aminotransferase. Males treated with 75 mg/kg bw/day also showed a statistically significant reduction in alanine aminotransferase. All of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral Assessments:
There were no treatment-related changes in the behavioral parameters at 15, 30 or 75 mg/kg bw/day.
One control female had noisy respiration recorded during behavioral assessments between Weeks 2 and 5 however this was considered to be incidental and a result of the method of administration.

Functional Performance Tests:
There were no toxicologically significant changes in functional performance at 15, 30 or 75 mg/kg bw/day.
Males from all treatment groups showed a statistically significant reduction in forelimb grip strength whilst females treated with 75 mg/kg bw/day showed a statistically significant increase in forelimb grip strength. The intergroup differences were confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments:
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 15, 30 or 75 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following microscopic abnormalities were detected:

Adrenals: Hypertrophy of the zona glomerulosa was present in two males and two females treated with 75 mg/kg bw/day, one female treated with 30 mg/kg bw/day and one male and one female treated with 15 mg/kg bw/day.

Kidneys: Increased hyaline droplets (mild) was evident in four males treated with 75 mg/kg bw/day. No such effects were detected in females treated with 75 mg/kg bw/day or in animals of either sex treated with 30 or 15 mg/kg bw/day.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating:
No treatment-related effects were detected in mating performance.

Fertility:
No treatment-related effects were detected in fertility.

Two females treated with 15 mg/kg bw/day and two females treated with 30 mg/kg bw/day were not pregnant following positive evidence of mating. No histopathological changes were evident in the female or male reproductive organs which could have been the cause of the infertility.

Gestation length:
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

One control female that had a total litter loss had a gestation length of 24½ days. It is not unusual for females with small litters and/or extended gestation periods to fail to maintain their litter. This was considered to be incidental as it was from a control group female and no similar effects in other groups.

Discussion:
The oral administration of Diallylamine to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of 15, 30 and 75 mg/kg bw/day did not result in any toxicologically significant effects.

Episodes of increased salivation were reported in animals of either sex treated with 75 mg/kg bw/day from Day 9 (males) and Day 13 (females) onwards. An increase in overall water consumption was also observed for females treated with 75 and 30 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are generally regarded as not an adverse effect of systemic toxicity. Initial reductions/losses in body weight gain were evident in animals of either sex treated with 75 mg/kg bw/day and in males treated with 30 mg/kg bw/day which was associated with reduced food consumption at 75 mg/kg bw/day, however subsequent recovery was evident in all animals and body weight gains which, in subsequent weeks for treated males were actually higher than control males. Due to the initial effect on body weight development in males treated with 75 mg/kg bw/day, overall body weight gains for this group was lower than controls. Based on the overall effect on body weight performance together with the clear evidence of recovery, these findings were deemed not to be of an adverse nature.

Although there were some statistically significant differences in treated animals from controls for the blood chemical parameters measured, in the absence of any related microscopic changes these differences were considered not to be of toxicological significance.

No treatment-related macroscopic abnormalities were evident in treated animals however microscopic examination revealed changes in the adrenal gland (hypertrophy of the zona glomerulosa) of animals of either sex treated with 75 and 15 mg/kg bw/day and in one female treated with 30 mg/kg bw/day. Zona glomerulosa hypertrophy in the adrenal gland is seen occasionally in toxicology studies and whilst the significance is not clear, it is linked to hydration and electrolyte balance and is therefore seen as an adaptive response. Due to the low incidence, lack of dose response and limited number of animals per group it is considered that, within the confines of this study the change may reflect a variation in background levels, particularly in lactating females and cannot be unequivocally related to the administration of the test item and not considered to be an adverse event.

Microscopic examination of the kidneys revealed increased hyaline droplets in males treated with 75 mg/kg bw/day. Kidney weights were also elevated in these males. Hyaline droplets can be directly linked to the accumulation of alpha 2u-globulin, which is unique to the male rat. This finding is not found in immature rats, female rats or humans and therefore is considered to be of no relevance to man. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for males can be established based on the observed effects excluding those related to alpha 2u-globulin nephropathy.

Mating performance and fertility was unaffected by treatment.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The oral administration of Diallylamine to rats by gavage, at dose levels of 15, 30 and 75 mg/kg bw/day, did not result in any significant toxicological effects.

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mating performance and fertility was unaffected by treatment.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistical analysis of the litter, offspring weights data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring:
Necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 15, 30 and 75 mg/kg bw/day.

Histopathological findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Statistical analysis of the surface righting reflex data did not reveal any significant intergroup differences.

In total eleven females from the control, ten females each from the 15 and 30 mg/kg bw/day dose groups and twelve females from the 75 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: there were no treatment-related effects detected in the offspring parameters observed.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The oral administration of Diallylamine to rats by gavage, at dose levels of 15, 30 and 75 mg/kg bw/day, did not result in any significant toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for either sex.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 75 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 15, 30 and 75 mg/kg bw/day. An additional male was included in the high dose group to replace a male which was terminated on Day 3 because this death was unrelated to treatment. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study (Day 17 for the additional high dose male and respective partner), with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 41 (additional high dose group male only), Day 43 or Day 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality

One male treated with 75 mg/kg bw/day was killedin extremison Day 3 due to a physical injury that was not related to treatment. This male was replaced on the study by Male No. 97. There were no further unscheduled deaths.

Clinical Observations

Animals of either sex treated with 75 mg/kg bw/day showed incidences of increased salivation from Week 2 onwards. No such effects were evident in animals of either sex treated with 30 or 15 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters at 15, 30 or 75 mg/kg bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 15, 30 or 75 mg/kg bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 15, 30 or 75 mg/kg bw/day.

Body Weight

Males treated with 75 and 30 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in the majority of males treated with 75 mg/kg bw/day and in three males treated with 30 mg/kg bw/day. Recovery was evident in males thereafter. Consequently overall body weight gain for males treated with 75 mg/kg bw/day was lower than controls. No such effects were detected in males treated with 15 mg/kg bw/day.

Females treated with 75 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in the majority of females. A slight reduction in body weight gain was also evident during the second week of maturation and during lactation. No such effects were detected in females treated with 30 or 15 mg/kg bw/day.

Food Consumption

Males treated with 75 mg/kg bw/day showed a reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. Food conversion efficiency was also reduced in these males and in males treated with 30 mg/kg bw/day during the first week of treatment. No such effects were detected in males treated with 15 mg/kg bw/day.

Females treated with 75 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency during both weeks of maturation. Food consumption was also reduced during the first week of gestation in these females. Recovery was evident thereafter. No such effects were detected in females treated with 30 or 15 mg/kg bw/day.

Water Consumption

Females treated with 75 and 30 mg/kg bw/day showed an increase in overall water consumption during maturation. No such effects were detected in females treated with 15 mg/kg bw/day or treated males.

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

No treatment-related effects were detected in fertility.

Gestation Lengths

Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum, sex ratio and viability were comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights on Days 1 and 4 post partum were comparable to control litters. Surface righting was also comparable to controls.

Laboratory Investigations

Hematology

There were no treatment-related effects detected in the hematological parameters examined.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Pathology

Necropsy

No toxicologically significant effects were detected in animals of either sex treated with 15, 30 and 75 mg/kg bw/day.

Organ Weights

Males treated with 75 mg/kg bw/day showed an increase in kidney weight both absolute and relative to terminal body weight. No toxicologically significant effects were detected in females treated with 75 mg/kg bw/day or in animals of either sex treated with 30 or 15 mg/kg bw/day.

Histopathology

The following microscopic abnormalities were detected:

Adrenals:Hypertrophy of the zona glomerulosa was present in two males and two females treated with 75 mg/kg bw/day, one female treated with 30 mg/kg bw/day and one male and one female treated with 15 mg/kg bw/day.

Kidneys:Increased hyaline droplets (mild) was evident in four males treated with 75 mg/kg bw/day. No such effects were detected in females treated with 75 mg/kg bw/day or in animals of either sex treated with 30 or 15 mg/kg bw/day.

Conclusion

The oral administration of Diallylamine to rats by gavage, at dose levels of 15, 30 and 75 mg/kg bw/day, did not result in any significant toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for either sex.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 75 mg/kg bw/day.

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

See details of the conducted OECD 422 study in the effects on fertility section.

Litter responses:

Offspring Litter Size, Sex Ratio and Viability:

No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Offspring Growth And Development:

There were no toxicologically significant effects detected.

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Necropsy:

Necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 15, 30 and 75 mg/kg bw/day.

There were no treatment-related effects detected in the offspring parameters observed.

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

An oral (gavage) combined repeat dose toxicity study with reproductive/developmental toxicity screening test in the rat (OECD 422) was conducted on the test substance.

The oral administration of Diallylamine to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of 15, 30 and 75 mg/kg bw/day did not result in any toxicologically significant effects.

Mating performance and fertility was unaffected by treatment and there were no treatment-related effects detected in the offspring parameters observed.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 75 mg/kg bw/day.

Based on the results of the OECD 422 study there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development at the highest dose level tested. The substance is therefore not classified for reproductive toxicity.