2-[(3-aminopropyl)methylamino]ethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-07-05 to 2019-11-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2019

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: (EC) No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(3-aminopropyl)methylamino]ethanol

EC Number:

255-615-9

EC Name:

2-[(3-aminopropyl)methylamino]ethanol

Cas Number:

41999-70-6

Molecular formula:

C6H16N2O

IUPAC Name:

2-[(3-aminopropyl)(methyl)amino]ethan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

- Name: APMMEA
- Physical state/appearance: clear, colorless liquid
- Batch number: PFW110370
- CAS number 41999-70-6
- Label: aminopropylmonomethylethanolamine (APMMEA) Lot no: PFW110370 (1.000 L) Containing 2-[3-aminopropyl)methylamino]ethanol
- Expiry date: 2018-12-31


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in darkness, may be used/formulated in light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar HanTM:RccHanTM:WIST strain

Details on species / strain selection:

The rat was selected 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: Envigo RMS (UK) Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males: Approximately 8 weeks on arrival, approximately 14 weeks at start of treatment
Females: Approximately 11 weeks on arrival, approximately 12 weeks at start of treatment
- Weight at study initiation: 289-351 g (males), 192-259g (females)
- Fasting period before study: no
- Housing:
During the pre-pairing phase: in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding (Dates and Ltd., Cheshire, UK).
During the pairing phase: Animals were paired on a one male: one female basis within each dose group in polypropylene cages with stainless steel mesh lids and grid flooring suspended over polypropylene trays lined with absorbent paper.
Following the pairing phase: Males were re-housed in their original pre-pairing cages. Mated females were individually housed in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding (Dates and Ltd., Cheshire, UK) for the gestation, birth and lactation phases of the study.
- Diet (e.g. ad libitum): ad libitum, free access to Rodent 2018C Teklad Global Certified Pelleted Diet
- Water (e.g. ad libitum): ad libitum, mains drinking water from polycarbonate bottles attached to the cages
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours of continuous low intensity fluorescent lighting in each 24 hour period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
- the test item was prepared at the appropriate concentrations as a solution in distilled water.
- formulations were prepared weekly for the first two weeks of treatment (as only 12 day stability had been confirmed), formulations were then prepared evey 2 weeks once 22 day stability data had been determined and stored at approx 4 °C in the dark.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):not specified
- Concentration in vehicle: 0 mg/mL, 20 mg/mL, 60 mg/mL, 120/90 mg/mL
- Amount of vehicle (if gavage): 5mL/kg. the volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
- Lot/batch no. (if required): not specified
- Purity:not specified

Details on mating procedure:

- M/F ratio per cage: one male: one female basis within each dose group
- Length of cohabitation: up to 14 days.
- Proof of pregnancy: 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.

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 part of parturition
iv. Date and time of observed completion of parturition

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity of the test item formulations were determined by the test facility. Results showed the formulations to be stable for at least 22 days.
- samples of test item formulations were taken on 3 occasions and analyzed for concentration of the test item.

Duration of treatment / exposure:

- males: approx 6 weeks
- females: 8 weeks (including a two-week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (low dose group)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 (intermediate dose group)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Group 4 (high dose group), until day 8

Doses / concentrations 5

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

group 4 (high dose group), from day 8 until end of the study

No. of animals per sex per dose:

24 animals (12 male and 12 female) per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (including a 14 day repeated dose oral (gavage) tange-finding toxicity study in the rat (Envigo Study Number: DG88JW)).
- Treatment volume: 5 mL/kg body weight
- Rationale for animal assignment: animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups
- Administration throughout the treatment was made using a stainless steel cannula attached to a disposable plastic syringe.
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
immediately before dosing, soon after dosing and one hour after dosing during the workweek

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
immediately before dosing, soon after dosing and one hour after dosing during the workweek
- Parameters: overt signs of toxicity, ill-health, behavioral change
- 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 a approx 0830 and as late as possible at weekends and public holidays. Date of apiring, date of mating, date and time of observed start of parturition, date and time of observed completion of parturition was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded on day 1 (prior to dosing) and then weekly for males until termination 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, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE:
yes
- Male dietary intake was recorded weekly prior to and after pairing.
- Female dietary intake was recorded weekly prior to pairing. 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 for the periods covering post partum days 1-4, 4-7 and 7-14.
- Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and females prior to pairing, and for males after the pairing phase.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Gravimetric measurement of water consumption was undertaken on all animals during the pre-pairing phase of the study.

HAEMATOLOGY: Yes
Hematological investigations were performed on five males and five females, selected from each dose group on Day 43 (males) and Day 13 postpartum (females). Blood samples were withdrawn from the lateral tail vein. Repeat blood samples were withdrawn by cardiac puncture at termination (Day 14) if required. Animals were not fasted prior to blood sampling.
- Parameters checked: Prothrombin time (CT), Platelet count (PLT), Activated partial thromboplastin time (APTT), Reticulocyte count (Retic), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct).

CLINICAL CHEMISTRY: Yes
Blood chemical investigations were performed on five males and five females, selected from each dose group on Day 43 (males) and Day 13 postpartum (females). Blood samples were withdrawn from the lateral tail vein. Repeat blood samples were withdrawn by cardiac puncture at termination (Day 14) if required. Animals were not fasted prior to blood sampling.
- Parameters checked: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili), Bile acids

OTHER:
FUNCTIONAL OBSERVATIONS:
- prior to start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on days 4, 11 and 18 post coitum and for littering females on days 4 and 12 post partum. 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 for each animal using a purpose built arena
- parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation

FUNCTIONAL PERFORMANCE:
- 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 approx the same time on each ocasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was 30 minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall 30 minute period and also during the final 20% of the period.
- 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 through 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.

SENSORY REACTIVITY:
- each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli
- parameters: grasp response, vocalization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex

THYROID HORMONE ANALYSIS
- serum and plasma samples were taken from all adult males and females at termination.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

PARAMETERS EXAMINED
- on completion of parturition (day 0 post partum), the number of live and dead offspring was recorded.
-The following parameters were examined in F1 offspring: Number of offspring born, Number of offspring alive (recorded daily and reported on days 1, 4, 7 and 13), Sex of offspring on Day 1, 4 , 7 and 13 postpartum, Clinical condition of offspring from birth to Day 13 postpartum, Individual offspring weights on Day 1, 4, 7 and 13 postpartum (litter weights were calculated retrospectively from this date)
- physical development: all live offspring were assessed for ano-genital distance on day 1 post partum. Visible nipple count was performed for all male offspring at Day 13 postpartum


THYROID HORMONE ANALYSIS
- where possible serum samples were taken from two randomly allocated offspring from each litter on day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on day 4 post partum.
- where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on day 13 post partum. Wher possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- All adult animals were normally killed by intravenous overdose of a suitable barbiturate agent, followed by exsanguination at termination.
- on day 44 or 45 (males), on day 14 post partum (females)

GROSS NECROPSY
- Post mortem procedures were performed on all adult animals and included any animal found dead or killed in extremis during the study.
- full external and internal examination

ORGAN WEIGHTS
- five males and five females from each dose group
- following organs were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (weihed partially fixed), Prostate, Seminal vesicles (with coagulation gland), spleen, Testes, Thymus, Thyroid (weighed partially fixed with parathyroid), Uterus (weighed with cervix and oviducts)

HISTOPATHOLOGY
- from five selected males and five selected females from each dose group and preserved in buffered 10% formalin
- tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin
- Tissues / organs collected: Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and Bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Cowpers glands, Duodenum, Esophagus, Eyes (retained in Davidson's Fluid), Glans penis, Gross lesions, Heart, Ileum (including peyer's patches), Jejunum, Kidneys, LABC (levator ani-bulbocavernous) muscle, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulation gland), Skin, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes (retained in Modified Davidson's Fluid), Thymus, Thyroid/Parathyroid, Trachea, Urinary bladder, Uterus and Cervix (with oviducts), Vagina

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation; blood samples were collected immediately following decapitation.

GROSS PATHOLOGY
All offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

Statistics:

Homogeneity of variance from mean values was analyzed using Bartlett's test.
Intergroup variance was assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates.
Any transformed data were analyzed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data.
If no dose response is found, but the data showed non­ homogeneity of means, the data was analyzed by a stepwise Dunnett's (parametric) or Steel (non-parametric) test to determine significant difference from the control group.
Where the data were unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett's test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett's test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann­ Whitney "U" test. Dose response relationships may also have been investigated by linear regression. Where the data was unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Alternatively, Fisher's exact test or chi-squared probability test may have been used where appropriate.

Reproductive indices:

Precoital interval: calculated as the time elapsing between initial pairing and the observation of positive evidence of mating
Mating index: (Number of animals mated / number of paired animals) x 100
Pregnancy index (%): (number of presumed pregnant animals/number of paired animals) x 100
Gestation length: calculated as the number of days of gestation includign the day for observation of mating and the start of parturition
Parturition index (%): (number of females delivering live offspring/number of pregnant females) x 100

Offspring viability indices:

Post - implantation loss (%): (number of implantations – total number of offspring born/ Number of implantation sites) x 100
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1)x 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) x 100
% Male offspring (Sex Ratio): calculated for each litter value on days 1, 4, 7 and 13 post partum = (Number of male offspring/ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 600/450 mg/kg bw/day showed instances of noisy respiration from Day 1 throughout the majority of the treatment period. Sporadic instances of increased salivation were noted in four males and eight females from Day 1 to Day 32 (males) and Day 5 to Day 32 (females).
One male animal exhibited hunched posture on Day 32 with one female exhibiting hunched posture and pilo-erection on Day 5 with hunched posture being apparent until Day 7.
Eight males and nine females animals treated with 300 mg/kg bw/day exhibited sporadic instances of noisy respiration from Days 4 to 37 (males) and from Days 5 to 52 (females).
Isolated occurrences of increased salivation (for one or two days only) were noted in one female and one male respectively. One female animal also exhibited hunched posture and staining around the mouth on Day 7. Small superficial scabs were also noted in one female animal from Days 30 to 33. As these observations were noted in isolation (in this treatment group) they were considered to be incidental and unrelated to treatment with the test item.
Increased salivation is commonly observed in this type of study and is generally considered to be due to an irritant/unpalatable nature of the test item and/or formulation. As such this observation is considered not to be specifically related to systemic toxicity of the test item.
Signs of noisy respiration may represent a potential difficulty in dosing these animals which may have caused a slight reflux of the test item or vehicle and cannot be considered to be related specifically to toxicity of the test item.
No clinical signs were apparent in any animals treated with 100 mg/kg bw/day.
Two control female animals exhibited noisy respiration on one or two days only.
The male animal that was humanely killed on Day 6 exhibited decreased respiratory rate, labored respiration, sneezing, hunched posture, pilo-erection and dehydration. Clinical signs of noisy respiration and increased salivation were noted from Day 2 and on Day 5 (respectively) prior to the female animal being found dead on Day 6.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal treated with 600 mg/kg bw/day was found dead on Day 6 and one male animal from this treatment group was humanely killed on Day 6 due to the severity of the observations noted. There were no further unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effect of treatment on body weight development in treated male animals once the dose level had been reduced to 450 mg/kg bw/day.
Males treated with 600 mg/kg bw/day exhibited reductions in body weight gain during the first week of the treatment period, however, recovery was evident thereafter (once the dose level had been reduced). These animals subsequently showed a statistically significant (p<0.01) increase in body weight gain during the third week of treatment. An increase in body weight gain is considered not to be an adverse effect of treatment and therefore is considered not to be toxicologically significant. An overall body weight gain for these animals still remained 5.4 % lower than control.
There was considered to be no obvious effects on body weight development in treated females during maturation.
Females treated with 600/450 mg/kg bw/day exhibited reductions in body weight gains throughout gestation when compared to control which achieved statistical significance from Days 14 – 20 (p<0.05). This resulted in a 19% reduction in overall body weight gain which achieved statistical significance (p<0.01), this may have been a consequence of the slightly reduced litter sizes at this dosage.
There was considered to be no adverse effect of treatment on body weight development in female animals treated with 100 and 300 mg/kg bw/day during gestation.
Slight reductions in body weight gains were noted during gestation for female animals treated with 100 or 300 mg/kg bw/day period, however, a dose relationship was generally not apparent and statistical significance was not achieved. Overall body weight gains during this period were approximately 10 % and 8 % lower than control for females animals treated with 100 and 300 mg/kg bw/day respectively.
There was considered to be no adverse effect of treatment on body weight development during lactation in any treated female. Female animals from all treatment groups exhibited slight reductions in body weight gains from Days 7 to 14, however, statistical significance was not achieved and a dose relationship was not apparent.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effects in food consumption in any treated male animals. A slight reduction in food consumption was noted in male animals treated with 600 mg/kg bw/day during the first week of treatment. However, recovery was evident thereafter such that food consumption was comparable to control throughout the remainder of the treatment period. The reduction noted may have been mainly due to the animal that was humanely killed on Day 6 and as such can be considered not to reflect this group as a whole.
No such effects were noted in male animals treated with 100 or 300 mg/kg bw/day.
There was considered to be no adverse effects noted in food consumption for treated females during maturation.
Slight reductions in food consumption were noted during the first week of treatment in animals treated with 600 and 300 mg/kg bw/day with recovery being noted during the second week of maturation. The reduction noted in animals treated with 600 mg/kg bw/day may have been mainly attributable to the animal that was found dead on Day 6 and as such can be considered not to reflect this group as a whole.
There was considered to be no adverse effects noted in food consumption for treated females during gestation.
Females treated with 600/450 mg/kg bw/day exhibited statistically significant reductions (p<0.05) in food consumption from Days 14 - 20. However, this could possibly be attributed to the lower litter sizes noted within this treatment group.
There were considered to be no adverse effects in food consumption during lactation for any treated female.
Slight reductions in food consumption were noted in females treated with 600/450 mg/kg bw/day throughout lactation but without achieving statistical significance. However, this could possibly be attributed to the lower litter sizes noted within this treatment group.
Slight reductions in food consumption were also noted in females treated with 300 mg/kg bw/day throughout lactation but without achieving statistical significance. This was considered to be mainly attributable to one female animal which was only maintaining 2 offspring for the majority of this phase of the study. With this animals data removed, group means were comparable to control.
No such effects were noted in female animals treated with 100 mg/kg bw/day.

Food efficiency:

no effects observed

Description (incidence and severity):

There was considered to be no adverse effects in food conversion efficiency in any treated male.
At 600/450 mg/kg bw/day, food conversion efficiency for males appeared slightly inferior to control from Days 1 to 8 and from Days 29 to 36.
Intergroup differences in food conversion efficiency for females during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 100, 300 or 600/450 mg/kg bw/day.

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

effects observed, treatment-related

Description (incidence and severity):

Male animals treated with 600/450 mg/kg bw/day exhibited general increases in water consumption throughout the pre-pairing phase of the study. There were considered to be no adverse effects in water consumption for any treated female animal.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the hematological parameters examined.
Statistically significant increases in erythrocytes (p<0.01) were noted in male animals treated with 600/450 mg/kg bw/day. All control animals exhibited values that were lower than the historical control data range whereas only one value was higher than historical control for the animals treated with 600/450 mg/kg bw/day. All treated males exhibited a statistically significant reduction (p<0.05) in mean corpuscular volume. With the exception of one value from animals treated with 300 mg/kg bw/day which was lower, all values were within the historical control data range. A statistically significant increase (p<0.01) in reticulocytes was also noted in male animals from all treatment groups, with a dose relationship being apparent.
Female animals treated with 600/450 mg/kg bw/day also exhibited a statistically significant increase (p<0.01) in reticulocytes. All values for reticulocytes were found to be within the normal historical control data range. As the majority of these values were found to be within the historical control data ranges and there were no histopathological correlates these findings are considered to be of no toxicological significance.
Female animals treated with 300 mg/kg bw/day exhibited a statistically significant decrease (p<0.01) in platelet count. All values for the treated animals were lower than the historical control data range. As similar effects were not apparent in female animals treated with 600/450 mg/kg bw/day this finding is considered to be incidental and unrelated to treatment with the test item.
No such findings were noted in female animals treated with 100 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were considered to be no toxicologically significant effects detected in the blood chemical parameters examined.
Male animals treated with 100 mg/kg bw/day exhibited a statistically significant increase (p<0.05) in potassium. All values were found to be within the historical control data range, the mean value for these animals was also lower than the mean values for males treated with 300 or 600/450 mg/kg bw/day and as such is considered to be of no toxicological significance.
Female animals treated with 600/450 mg/kg bw/day exhibited statistically significant decreases in phosphorous (p<0.01). One control value was much higher than the historical control data range, whereas all values for the treated animals were within the historical control data range, a true dose relationship was not apparent.
No such findings were noted in any treated male animal or in female animals treated with 100 or 300 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral assessments: A small number of instances of noisy respiration were noted during the behavioral assessments in male animals from all treatment groups with increased occurrences with increases in dose level. An isolated occurrence of chromodacryorrhea was also noted in one male animal treated with 600/450 mg/kg bw/day. Isolated occurrences of noisy respiration were also noted in female animals from control, 300 or 600/450 mg/kg bw/day.
There were no changes in the behavioural parameters in female animals treated with 100 mg/kg bw/day.

Functional performance tests: There were considered to be no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 600/450 mg/kg bw/day. Male animals treated with 300 mg/kg bw/day exhibited a statistically significantly higher (p<0.05) hind limb grip strength. As the increase in hind limb grip strength was only noted in one out of the three runs completed and there were no clinical signs observed to signify a neurotoxic effect of the test item this was considered to be incidental and of no toxicological relevance. Female animals from all treatment groups exhibited a statistically significant increase (p<0.05) in overall activity. As a dose relationship was not apparent and there were no clinical signs observed to signify a neurotoxic effect of the test item these were considered to be incidental and of no toxicological relevance.

- sensory reactivity: there were no inter-group differences in sensory reactivity scores at 100, 300 or 600/450 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):

- Premature decedents: There were 2 premature decedent animals treated with 600 mg/kg bw/day which were found dead or killed in extremis prior to the reduction in dose on Day 8.
One male animal was killed in extremis on day 6. At histopathology examination in the stomach there was ulceration and erosion of the glandular mucosa and hyperplasia of the nonglandular area. These changes are considered likely to have contributed to the poor condition of the animal. There were also stress related changes with adrenal cortical hypertrophy and thymic atrophy. There was degranulation in the pancreas, likely to indicate reduced food intake.
The female which was found dead on Day 6 only had one notable change at histopathological examination which was marked necrosis of the tracheal epithelium which was considered to be the cause of death.
- terminal kill:
- Adrenal Glands
Hypertrophy of the zona glomerulosa, at minimal severity, was noted in 2/5 females treated with 100 mg/kg bw/day and 4/5 females treated with 300 mg/kg bw/day or 600/450 mg/kg bw/day. This is considered likely to be an adaptive response.
- Stomach
Erosion was present in the glandular region of one male and one female treated with 600/450 mg/kg bw/day. Even though these were of low incidence they were considered to be adverse.
- Trachea
Ulceration of the trachea was present in one male treated with 600/450 mg/kg bw/day. This animal also had inflammatory change in the lungs, including exudate in the airways. One female treated with 300 mg/kg bw/day had ulceration in the trachea along with metaplasia (regeneration). In both the early decedents and animals dosed at the reduced level of 450 mg/kg bw/day, the histopathological findings in the trachea of some animals, are indicative of blockage caused by reflux due to gavage dosing of an irritant (Damsch 2011). No changes were present in the trachea of animals treated with 100 mg/kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analysis: Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 600/450 mg/kg bw/day.
A statistically significant increase (p<0.05) in Thyroxine (T4) was noted in male offspring from females treated with 300 mg/kg bw/day. As no such effects were noted in female offspring or from offspring of either sex at the higher dosage, this was considered to be incidental and unrelated to treatment with the test item.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle. With the exception of one female from each of the treatment groups, all females showed regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrous on the day of necropsy.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic findings noted in the reproductive tracts 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):

Fertility
No treatment-related effects were detected in fertility. One female treated with 600/450 mg/kg bw/day and one treated with 300 mg/kg bw/day were non-pregnant following positive evidence of mating. One female treated with 600/450 mg/kg bw/day was found dead prior to pairing. Two females treated with 100 mg/kg bw/day were non-pregnant or did not have a litter following positive evidence of mating.
Gestation Length
Gestation lengths for controls and treated females were between 22 and 24 days. Overall, the distribution of gestation lengths for treated females were essentially similar to control.
Litter Responses
There were 0, 2, 1 and 1 females (that survived to parturition) that failed to achieve pregnancy in the control, 100, 300 and 600/450 mg/kg bw/day dosage groups respectively. The following assessment is generally made using the 12, 10, 11 and 10 litters which were reared to Day 13 of age.
Offspring Litter Size, Sex Ratio and Viability
There was considered to be no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 100, 300 or 600/450 mg/kg bw/day. At 600/450 mg/kg bw/day, the mean number of implantations was lower than control although this value failed to attain statistical significance. The number of implantations was not particularly low at this dosage and this difference appears to be incidental and unrelated to maternal treatment. With the exception of one value (this animal exhibited a unilateral pregnancy which was considered to be incidental and un-related to treatment) which was slightly lower, all implantation values were withi n the normal historical control data range. The mean total number of offspring born was slightly lo wer than control but consistent with the previous slightly lower mean number of implantations at this dosage. Litter size on Days 1, 4, 7 and 13 post partum were also slightly lower for females treated with 600/450 mg/kg bw/day when compared to control litters which again failed to achieve statistical significance. Live birth index and offspring viability in treated females was comparable to controls. Sex ratio across all treatment groups was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.

Details on results (P0)

One female animal treated with 600 mg/kg bw/day was found dead on Day 6 and one male animal from this treatment group was humanely killed on Day 6 due to the severity of the observations noted. There were no further unscheduled deaths during the study.
Animals of either sex treated with 600/450 mg/kg bw/day showed instances of noisy respiration throughout the majority of the treatment period. Sporadic instances of increased salivation were noted in four males and eight females. One male animal exhibited hunched posture for one day, one female exhibited pilo-erection for one day and hunched posture for three days.
Eight males and nine female animals treated with 300 mg/kg bw/day exhibited sporadic instances of noisy respiration. Isolated occurrences of increased salivation were noted in one male and one female. One female animal also exhibited hunched posture and staining around the mouth for one day.
No clinical signs were apparent in any animals treated with 100 mg/kg bw/day.
The male animal that was humanely killed on Day 6 exhibited decreased respiratory rate, labored respiration, sneezing, hunched posture, pilo-erection and dehydration. Clinical signs of noisy respiration and increased salivation were noted from Day 2 and on Day 5 (respectively) prior to the female animal being found dead on Day 6.
A small number of instances of noisy respiration were noted during the behavioral assessments in male animals from all treatment groups with increased occurrences with increases in dose level. An isolated occurrence of chromodacryorrhea was also noted in one male animal treated with 600/450 mg/kg bw/day. Isolated occurrences of noisy respiration were also noted in female animals from control, 300 or 600/450 mg/kg bw/day.
There were no changes in the behavioral parameters for female animals treated with 100 mg/kg bw/day.
There were considered to be no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 600/450 mg/kg bw/day.
There were no inter-group differences in sensory reactivity scores at 100, 300 or 600/450 mg/kg bw/day. Male animals treated with 600 mg/kg bw/day exhibited a reduction in body weight gain during the first week of treatment. Recovery was evident once the dose level had been reduced to 450 mg/kg bw/ day, there was subsequently no adverse effect of treatment on body weight development.
There was considered to be no obvious effects on body weight development in treated females during maturation.
Females treated with 600/450 mg/kg bw/day exhibited reductions in body weight gains throughout gestation when compared to control. This resulted in an overall reduction in body weight gain, this may reflect the lower litter sizes at this dosage.
There was considered to be no adverse effect of treatment on body weight development in female animals treated with 100 and 300 mg/kg bw/day during gestation.
There was considered to be no adverse effect of treatment on body weight development during lactation in any treated female.
There was considered to be no adverse effects in food consumption in any treated males or in females during maturation, gestation or lactation.
There was considered to be no adverse effects in food conversion efficiency in any treated males or in females during the pre-pairing phase of the study.
Male animals treated with 600/450 mg/kg bw/day exhibited general increases in water consumption throughout the pre-pairing phase of the study. There were considered to be no adverse effects in water consumption for any treated female animal.
Reproductive results
Mating- No treatment-related effects were detected in mating performance.
Fertility - No treatment-related effects were detected in fertility. One female treated with 600/450 mg/kg bw/day and one treated with 300 mg/kg bw/day were non-pregnant following positive evidence of mating. One female treated with 600/450 mg/kg bw/day was found dead prior to pairing. Two females treated with 100 mg/kg bw/day were non-pregnant or did not have a litter following positive evidence of mating.
Gestation Length - Gestation lengths for controls and treated females were between 22 and 24 days. Overall, the distribution of gestation lengths for treated females were essentially similar to control.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the findings in general do not reflect true systemic toxicity as the effects noted are consistent to be in relation to the irritant nature of the test substance

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical signs apparent for offspring on the study were typical for the age observed.
Neither the incidence nor distribution of these observations indicated any effect of maternal treatment on offspring development in any treatment group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Offspring viability in treated females was comparable to controls.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effect of treatment with the test item indicated by offspring body weight, body weight gain or litter weights.
As a consequence of the slightly reduced litter size from animals treated with 600/450 mg/kg bw/day, litter weights on Days 1, 4, 7, and 13 post partum were slightly reduced when compared to control, but without achieving statistical significance. However, offspring body weights were higher than control throughout the lactation phase of the study, with female offspring body weights achieving statistical significance (p<0.05) on Days 4 and 7 when compared to control. Offspring body weight gains on Days 1, 4, 7, and 13 post partum exceeded control litters but without achieving statistical significance.
No such effects were noted in offspring from animals treated with 100 or 300 mg/kg bw/day.

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:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase (p<0.05) in Thyroxine (T4) was noted in male offspring from females treated with 300 mg/kg bw/day. As no such effects were noted in female
offspring or from offspring of either sex at the higher dosage, this was considered to be incidental and unrelated to treatment with the test item.

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):

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 in any treatment group.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Offspring Litter Size, Sex Ratio and Viability
There was considered to be no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 100, 300 or 600/450 mg/kg bw/day. At 600/450 mg/kg bw/day, the mean number of implantations was lower than control although this value failed to attain statistical significance. The number of implantations was not particularly low at this dosage and this difference appears to be incidental and unrelated to maternal treatment. With the exception of one value (this animal exhibited a unilateral pregnancy which was considered to be incidental and un-related to treatment) which was slightly lower, all implantation values were within the normal historical control data range. The mean total number of offspring born was slightly lower than control but consistent with the previous slightly lower mean number of implantations at this do sage. Litter size on Days 1, 4, 7 and 13 post partum were also slightly lower for females treated with 600/450 mg/kg bw/day when compared to control litters which again failed to achieve statistical signifi cance. Live birth index and offspring viability in treated females was comparable to controls. Sex ratio across all treatment groups was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

- there were 0, 2, 1 and 1 females (that survived parturition) that failed to achieve pregnancy in the control, 100, 300 and 600/450 mg/kg bw/day dosage groups respectively. The following assessment is generally made using 12, 10, 11 and 10 litters which were reared to day 13 of age.
- there was considered to be no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to day 13 of age at any dose level.
- at 600/450 mg/kg bw/day, the mean number of implantations was lower than control although this value failed to attain statistical significance. THe number of implantations was not particularly low at this dosage and this difference appears to be incidental and unrelated to maternal treatment. With the exception of one value which was slightly lower, all implantation values were within the normal historical control data range. The mean total number of offspring born was slightly lower than control but consistent with the previous slightly lower mean number of implantations at this dosage. Litter size on days 1, 4, 7 and 13 post partum were also slightly lower for females treated with 600/450 mg/kg bw/day whn compared to control litters which again failed to achieve statistical significance.
- live birth index and offspring viability in treated females was comparable to controls
- sex ratio across all treatment groups was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.
- there was no effect of treatment with the test item indicated by ano-genital distance on day 1 post partum or visible nipple count in male offspring onday 13 post partum from any treatment group
- evaluation of thyroxine in offspring at day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 600/450 mg/kg bw/day. A statistically significant increase (p<0.05) in thyroxine (T4) was noted in male offspring from females treated with 300 mg/kg bw/day. As no such effects were noted in female offspring or from offspring of either sex at the higher dosage, this was considered to be incidental and unrelated to treatment with the test item.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical verification

The results indicate that the prepared formulations were within +/-10% of the nominal concentration.

Summary of results from the 14 day repeated dose oral (gavage) range-finding toxicity study in the rat:

Administration of APMMEA to Wistar Hanä:RccHanä:WIST strain rats for fourteen consecutive days at dose levels of up to 500 mg/kg bw/day (500, 300 and 100 mg/kg bw/day)resulted in reductions in body weight gains during the first week of treatment in animals of either sex from the high dose group. The effect in male animals was considered to be overemphasised by one male animals which exhibited body weight losses during the first week of treatment. Recovery was evident in both sexes during the second week of treatment such that male animals exhibited overall body weight gains which were comparable to control, whereas, female animals didn’t exhibit such a marked recovery and subsequently exhibited a 25% reduction in body weight gain when compared to control. No such effects were evident in the other two treated dose groups.

There was considered to be no adverse effect of treatment on food consumptions for any treated group when compared to controls and any marginal effects were considered to be reflective of the body weight losses noted at the high treatment level. 

It can be concluded that there was an apparent effect on body weight in animals of either sex treated with 500 mg/kg bw/day, however, there was only marginal effects on the corresponding food consumptions which were not generally considered to be adverse in nature. Improvements were generally noted after the first week of dosing suggesting that animals are adapting to the test item over time.

Taking into consideration the overall results from this study, a high dose level of 600 mg/kg bw/day is considered suitable for use in a subsequent Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) (Envigo Study Number: TX10VW). Furthermore, dose levels of 100 and 300 mg/kg bw/day are considered suitable as low and intermediate doses, respectively.

Tabular summary report of effects on reproduction/development

Observations		Dose level (mg/kg bw/day)
		0 (control)	100	300	600/450
paired animals	n	12	12	12	11
females showing evidence of copulation	n	12	12	12	11
pregnant females	n	12	11	11	10
conception days 1 -4	n	12	11	11	10
gestation = 22 days	n	3	3	1	2
gestation = 22.5 days	n	5	3	3	0
gestation = 23 days	n	4	2	6	7
gestation = 23.5 days	n	0	1	1	1
gestation = 24 days	n	0	1	0	0
dams with live young born	n	12	10	11	10
dams with live young at day 13 pp	n	12	10	11	10
implants/dam	x	13.4	11.7	12.7	10.9
live offspring/dam at day 1 pp	x	12.0	11.2	11.5	9.9
live offspring/dam at day 4 BC pp	x	11.8	11.1	10.9	9.6
live offspring/dam at day 4 AC pp	x	10.1	9.4	9.4	8.2
live offspring/dam at day 7 pp	x	10.1	9.4	9.4	8.1
live offspring/dam at day 13 pp	x	9.8	9.4	9.4	8.1
sex ratio: % males at day 1 pp	x	57.7	51.2	43.9	45
sex ratio: % males at day 4 BC pp	x	58.3	51.7	44.4	45.7
sex ratio: % males at day 4 AC pp	x	63.6	52.9	50.7	49.4
sex ratio: % males at day 7 pp	x	63.6	52.9	50.7	49.2
sex ratio: % males at day 13 pp	x	63.4	52.9	50.7	49.2
litter weight (g) at day 1 pp	x	68.06	63.29	67.35	60.12
litter weight (g) at day 4 BC pp	x	95.18	92.93	92.62	86.91
litter weight (g) at day 4 AC pp	x	81.15	78.93	79.71	74.51
litter weight (g) at day 7 pp	x	125.66	124.96	124.19	113.99
litter weight (g) at day 13 pp	x	244.84	246.18	241.50	220.58
male offspring weight (g) at day 1 pp	x	5.87	5.87	6.13	6.18
male offspring weight (g) at day 4 BC pp	x	8.22	8.67	8.88	9.15
male offspring weight (g) at day 4 AC pp	x	8.22	8.70	8.88	9.16
male offspring weight (g) at day 7 pp	x	12.73	13.62	13.70	14.13
male offspring weight (g) at day 13 pp	x	25.70	26.57	26.68	27.33
female offspring weight (g) at day 1 pp	x	5.49	5.61	5.71	6.06
female offspring weight (g) at day 4 BC pp	x	7.94	8.19	8.42	9.09
female offspring weight (g) at day 4 AC pp	x	7.89	8.21	8.38	9.06
female offspring weight (g) at day 7 pp	x	12.23	12.94	13.07	14.06
female offspring weight (g) at day 13 pp	x	24.79	25.96	25.67	27.23
Loss of offspring/dam
prenatal (implantations - live births)
0	n	3	6	5	3
1	n	4	3	2	5
2	n	4	1	2	1
3	n	0	0	1	1
4	n	0	0	1	0
5	n	1	0	0	0
post-natal (live births - offspring alive on day 13 pp) (excluding offspring culled on day 4 pp)
0	n	10	9	10	7
1	n	1	1	0	2
2	n	0	0	0	1
3	n	1	0	0	0
7	n	0	0	1	0

pp: post partum; n: number; x: mean

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance to Wistar Han™:RccHan™:WIST strain rats for a period of up to six weeks for males and eight weeks for females (including two weeks prepairing, gestation and early lactation for females) at dose levels of 100, 300 and 600 mg/kg bw/day resulted in the early deaths/terminations of one male and one female animal treated with 600 mg/kg bw/day.
This dose level was reduced to 450 mg/kg bw/day on Day 8. The primary cause of death in these animals was considered to be local toxicity. In both the early decedents and animals dosed at the reduced level of 450 mg/kg bw/day, the noisy respiration exhibited clinically, along with the histopathological findings in the trachea of some animals, are indicative of blockage caused by reflux due to gavage dosing of an irritant (Damsch 2011).
In general, it is considered that the effects noted in the animals at 600/450 mg/kg bw/day were considered to be the result of irritancy caused by the test item rather than attributable to true systemic toxicity.
A ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 450 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity as the effects noted are considered to be in relation to the irritant nature of the test item.
The `No Observed Adverse Effect Level' (NOAEL) for reproductive and developmental toxicity was considered to be 450 mg/kg bw/day.
The test substance is not to be classified as reproductive toxicant according to CLP Regulation.