[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

Endpoint summary

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD Guideline 422, the NOAEL (no observed adverse effect level) for general, systemic toxicity of (+)-3-Aminomethylpinan was the mid dose of 25 mg/kg bw/d. At the highest tested dose of 75 mg/kg bw/d, treatment-related adverse effects were observed based on a reduction in food consumption and a decrease in body weight (change) of male and female F0 parental animals. The NOAEL for reproductive performance and fertility of the F0 parental animals was the highest tested dose of 75 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 offspring was 25 mg/kg bw/d due to a delay in body weight development of F1 male and female pups at 75 mg/kg bw/d (BASF SE, 2018).

In a 15 d range-finding study in rats (BASF SE, 2016), a NOAEL of 50 mg/kg bw/d was determined.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

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)

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Version / remarks:

2000-07

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: WE 20PA15
- Expiration date of the batch: 2017-11-26

PURITY
Sum or all relevant peaks: 99.6%

CONTENT:
Main component nd diastereomers: > 98 g/100g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS:
- Liquid / colorless, clear

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guidelines require the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
(P) males: 11 - 12 weeks; females: 10 weeks
- Weight at study initiation:
(P) Males: 357.7 - 414.5 g; Females: 193.6 - 226.3 g;
- Housing: During pre-treatment of the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp). During the study period, the rats were housed individually in Polycarbonate cages type III with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
- Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Diet: Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: at least 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % with 5 mg/100 mL Tween 80 suspension in drinking water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance was weighed, topped up with 0.5 % Sodium carboxymethyl cellulose with 5 mg/100 mL Tween 80 suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Homogeneous distribution of the test substance in the vehicle
- Concentration in vehicle: 0.5 %
- Amount of vehicle: 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance concentrations were determined via GC (Shimadzu 2010 plus, FID, Dionex Chromeleon Software (dionex), or equivalent system). Based on the analytical results it is concluded that the test substance is stable in 0.5 % carboxymethyl cellulose in drinking water + Tween 80 (5 mg/100 mL) over a period of 7 days at a target concentration of 50 mg/100 mL at ambient temperature and over a period of 4 days at a target concentration of 1000 mg/100 mL also at ambient temperature. All determined concentrations were in the range of 90 % - 110 % of the nominal concentration.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, one day post-mating in males (15 d), and the entire gestation period as well as up to 22 days of the lactation period in females (37 d).

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Sponsors request
- Rationale for animal assignment: The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day (male and female animals) before the beginning of the administration period (day -1).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined once a week for male and female parental animals. Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20. Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13. Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from F0 parental male animals of all test groups shortly before sacrifice and from F0 parental female animals of all test groups on PND 14.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 16 - 20 h
- How many animals: The first 5 surviving parental males per group at termination and first 5 females with litters (in order of delivery) per group at PND 14.
- Following parameters were examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT), blood smears

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from F0 parental male animals of all test groups shortly before sacrifice and from F0 parental female animals of all test groups on PND 14.
- Animals fasted: Yes, 16 - 20 h
- How many animals: The first 5 surviving parental males per group at termination and first 5 females with litters (in order of delivery) per group at PND 14.
- Following parameters were examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Total bilirubin, Total Protein, Albumin, Globulins, Triglycerides, Cholesterol, Bile acid, Thyroid hormones (T4)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested:
Home cage observations: Posture, Tremors, Convulsions, Abnormal movements, Gait
Open field observations: Behavior on removal from cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Gait, Activity/arousal level, feces (consistency/color) within 2 minutes, Urine (amount/color) within 2 minutes, Rearing within 2 minutes
Sensory motor tests/Reflexes: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Motor activity measurement

IMMUNOLOGY: No

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, gross pathological assessment of all animals, with special attention being given to the reproductive organs.

HISTOPATHOLOGY: Yes (see table 1)

Other examinations:

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Bulbourethral gland (Cowper’s gland)
3. Epididymides
4. Glans penis
5. M. levator ani together with musc. bulbocavernosus (males only)
6. Prostate
7. Seminal vesicles with coagulating glands
8. Testes
9. Thyroid glands (fixed)

The following weights were determined in 5 animals per sex/test group sacrificed on schedule
(females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

Statistics:

DUNETT-test (two-sided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Up to four mid-dose (25 mg/kg bw/d) and all high-dose (75 mg/kg bw/d) male animals showed slight to severe salivation after treatment during several parts of the premating, mating and post-mating periods.
Up to eight high-dose female animals showed moderate salivation after treatment during premating and mating periods, while during gestation and lactation periods a maximum of five mid-dose females and all high-dose females (grade: slight to severe) showed this finding after treatment.
This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract.
It is not considered to be a sign of systemic toxicity. No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups during the entire study period.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight and body weight change of the mid- and low-dose F0 females and males (25 or 5 mg/kg bw/d) were comparable to the concurrent control group during the entire study period. The body weight change of the high-dose male animals (75 mg/kg bw/d) was statistically significantly decreased during premating days 0-7 (-85 % below control) and, consequently, if calculated for the entire premating period (days 0-13; -49 %). During mating, the body weight change of these animals was slightly reduced without attaining statistical significance (-24 %). This was assessed as treatment-related and adverse.
High dose female animlas showed mean body weight (change) values comparable to controls during premating, mating and gestation. During lactation (PND 0-4), females of test group 3 showed a body weight loss and a decrease of body weight change (-5.8 g) without statistical significance. They recovered and gained weight from PND 4 onwards. Although body weight was only affected in one study period during lactation, the significant body weight loss in the male animals was assessed as treatment-related and adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose F0 males (75 mg/kg bw/d) was statistically significantly below the concurrent control values during premating days 0-7 (around -10 %) and, consequently, if calculated for the entire premating period (days 0-13; around -8 %). This was assessed as treatment-related and adverse. Food consumption of the high-dose F0 females was also statistically significantly reduced during premating days 0-7 (around -8 % below control) and PND 4-7 (around -11 %). This was assessed as treatment-related and adverse. During gestation period, food consumption of the females in test group 3 was comparable to the concurrent control group. The mid- and low-dose F0 parental animals (25 and 5 mg/kg bw/d) did not show any test substance-related changes in food consumption during the entire treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. In males of test group 3 (75 mg/kg bw/d) hematocrit values were significantly lower compared to controls. However, the mean was within the historical control range (males, hematocrit 0.408-0.450 L/L). Therefore, this change was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed. In males of test group 3 (75 mg/kg bw/d) urea, triglyceride and inorganic phosphate levels were significantly increased. Urea and inorganic phosphate levels were within, triglyceride levels marginally above the historical control range (males, urea 3.93-7.15 mmol/L, inorganic phosphate 1.45-1.98 mmol/L, triglycerides 0.40-1.06 mmol/L). However, higher triglyceride values were the only changed parameter values among these individuals and therefore, this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No.85, 2002).
In females of test group 3 (75 mg/kg bw/d) total protein and globulin values were significantly decreased. Both mentioned protein means were slightly below whereas the albumin mean was within the historical control range (females, total protein 58.64-62.26 g/L, globulins 23.40-27.60 g/L; albumin 32.97-36.87 g/L). Total proteins consist of albumin and globulin fractions. Therefore, the decreased globulin levels resulted in decreased total protein levels when albumin values were not changed. The changed globulin levels were the only changed clinical pathology parameter in females of test group 3. Therefore, this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No.85, 2002).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared to control group 0 (set to 100%), the mean relative weights of liver and epididymides in males were significantly changed. All other mean relative weight parameters did not show significant differences when compared to the control group. The significant absolute weight increase of the epididymides in test group 1 occurred without dose-dependency and was incidental. The significant relative weight increases were all within the historical control range (0.272 – 0.342 %). Furthermore, no histopathological correlate to the weight increase was found. Therefore, the significant relative weight increases of the epididymides were al regarded as incidental and not treatment-related. The significant relative weight increase of the liver (2.537 %) in males was slightly above the historical control range (2.126 – 2.356 %) and was regarded as treatment-related but not adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related findings in male and female animals of all test groups in the sensorimotor tests.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minimal to slight centrilobular vacuolation of macrovesicular type in 5 out of 10 males was positive in the Oil-Red-O stain, which was consistent with a centrilobular fatty change. Furthermore, 3 out of 10 males showed a minimal centrilobular hypertrophy. These findings were regarded as treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In the ovaries of control and high dose females the different stages of functional bodies (especially corpora lutea) were present and normal.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones
In parental males (test groups 1, 2 and 3; 5, 25 and 75 mg/kg bw/d) and in male and female pups at PND 13 (test groups 11, 12 and 13; 5, 25 and 75 mg/kg bw/d), no treatment-related alterations of T4 levels (thyroid hormones) were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Critical effects observed:

no

Table 1: Summary Food Consumption Per Animal And Day

		Test Group 0 0 mg/kg bw/d	Test Group 1 5 mg/kg bw/d	Test Group 2 25 mg/kg bw/d	Test Group 3 75mg/kg bw/d
Male, premating
d0 - 7	Mean [g]	21.8 n	21.6	20.9	19.6 **
	S.D.	1.5	1.6	1.0	1.1
	Deviation vs Control [%]		-0.8	-4.2	-9.9
d7 - 13	Mean [g]	22.3 n	22.2	22.1	21.1
	S.D.	1.1	2.0	1.1	0.8
	Deviation vs Control [%]		-0.4	-0.9	-5.2
d0 - 13	Mean [g]	22.0 n	21.9	21.4	20.3*
	S.D.	1.3	1.7	1.0	0.9
	Deviation vs Control [%]		-0.6	-2.7	-7.0
Female, premating
d0 - 7	Mean [g]	14.1 n	14.4	14.2	13.0 *
	S.D.	0.7	0.7	1.1	0.9
	Deviation vs Control [%]		2.0	0.5	-8.1
d7 - 13	Mean [g]	14.4 n	14.8	14.8	14.5
	S.D.	0.9	0.7	1.4	1.1
	No of animals	10	10	10	10
	Deviation vs Control [%]		2.7	2.3	0.5
d0 - 13	Mean [g]	14.3 n	14.6	14.4	13.7
	S.D.	0.7	0.7	1.2	1.0
	No of animals	10	10	10	10
	Deviation vs Control [%]		2.3	1.3	-4.1
Female, Gestation
d0 - 7	Mean [g]	18.4 n	18.9	19.1	18.5
	S.D.	1.7	1.1	2.1	2.0
	Deviation vs Control [%]		2.3	3.8	0.5
d7 - 14	Mean [g]	21.2 n	20.2	21.2	19.7
	S.D.	2.6	1.5	2.5	1.3
	Deviation vs Control [%]		-4.7	0.1	-7.2
d14 - 20	Mean [g]	21.5 n	21.5	22.2	21.1
	S.D.	2.1	0.9	2.5	1.1
	Deviation vs Control [%]		-0.1	3.3	-2.2
d0 - 20	Mean [g]	20.3 n	20.1	20.8	19.7
	S.D.	2.0	1.1	2.1	1.1
	Deviation vs Control [%]		-1.019.1	2.3	-3.2
Female, Lactation
d1 - 4	Mean [g]	29.1 n	31.8	27.7	25.2
	S.D.	3.6	3.9	5.4	4.4
	Deviation vs Control [%]		9.3	-4.9	-13.6
d4 – 7	Mean [g]	38.1 n	40.4	37.1	34.1 *
	S.D.	2.4	3.4	3.9	3.7
	Deviation vs Control [%]		6.1	-2.5	-10.6
d7 - 10	Mean [g]	46.1 n	48.7	45.5	42.1
	S.D.	2.9	4.7	3.9	3.1
	Deviation vs Control [%]		5.6	-1.4	-8.7
d10 - 13	Mean [g]	55.2 n	55.8	52.9	51.2
	S.D.	3.6	3.9	5.1	3.3
	Deviation vs Control [%]		1.0	-4.2	-7.4
d1 - 13	Mean [g]	42.1 n	44.2	40.8	38.3
	S.D.	2.6	3.6	4.2	3.3
	Deviation vs Control [%]		4.9	-3.1	-9.0
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, d = day; n=DUNNETT

Table 2: Summary Changes Body Weights

		Test Group 0 0 mg/kg bw/d	Test Group 1 5 mg/kg bw/d	Test Group 2 25 mg/kg bw/d	Test Group 3 75mg/kg bw/d
Male, premating
d0 - 7	Mean [g]	11.8 n	12.0	8.1	1.8 **
	S.D.	5.2	6.5	4.8	4.8
d7 - 13	Mean [g]	13.1 n	9.8	12.2	11.0
	S.D.	5.1	6.3	5.2	5.4
d0 - 13	Mean [g]	25.0 n	21.8	20.3	12.8 **
	S.D.	6.9	11.0	6.4	7.9
Male, mating
d7 - 14	Mean [g]	10.8 n	10.4	11.1	8.2
	S.D.	4.4	3.0	4.4	2.7
Female, premating
d0 - 7	Mean [g]	-0.8 n	0.5	5.2	1.1
	S.D.	9.3	6.6	7.5	5.9
d7 - 13	Mean [g]	6.9 n	3.5	2.4	3.7
	S.D.	5.5	7.5	6.9	6.1
d0 - 13	Mean [g]	6.1 n	4.0	7.7	4.8
	S.D.	5.2	4.0	6.0	3.9
Female, gestation
d0 - 7	Mean [g]	26.3 n	26.6	29.8	25.4
	S.D.	6.5	5.	7.1	5.0
d7 - 14	Mean [g]	30.6 n	27.3	26.6	29.8
	S.D.	4.8	3.7	6.4	5.0
d14 - 20	Mean [g]	58.2 n	60.8	51.4	55.7
	S.D.	10.1	8.9	20.6	10.7
d0 - 20	Mean [g]	115.1 n	114.7	107.8	111.0
	S.D.	16.4	8.6	28.6	14.5
Female, lactation
d1 - 4	Mean [g]	3.9 n	7.3	2.7	-5.8
	S.D.	7.8	13.4	12.0	10.9
d4 – 7	Mean [g]	4.5 n	9.4	7.7	9.6
	S.D.	7.6	8.2	2.6	7.6
d7 - 10	Mean [g]	12.9 n	10.5	9.7	11.9
	S.D.	7.4	4.2	5.4	5.0
d10 - 13	Mean [g]	4.8 n	3.0	5.0	7.7
	S.D.	4.9	7.0	6.2	7.5
d1 - 13	Mean [g]	26.1 n	30.1	25.2	23.4
	S.D.	8.6	12.9	10.0	6.7
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, d = day; n=DUNNETT

Conclusions:

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of (+)-3-Aminomethylpinan was the mid dose of 25 mg/kg bw/d. At the highest tested dose of 75 mg/kg bw/d, treatment-related adverse effects were observed based on a reduction in food consumption and a decrease in body weight (change) of male and female F0 parental animals. The NOAEL for reproductive performance and fertility of the F0 parental animals was the highest tested dose of 75 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 offspring was 25 mg/kg bw/d due to a delay in body weight development of F1 male and female pups at 75 mg/kg bw/d.

Executive summary:

(+)-3-Aminomethylpinan was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 5, 25 and 75 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose with 5 mg/100 ml Tween 80 suspension in drinking water). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, one day post-mating in males, and the entire gestation period as well as up to 22 days of the lactation period in females. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams, food consumption was determined for gestation days (GD) 0-7, 7-14, 14-20 and postnatal days (PND) 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 4 and 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The following test substance-related adverse effects/findings were noted:

Test group 3 (75 mg/kg bw/d):

F0 PARENTAL ANIMALS / CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

F0 males:

- Statistically significantly reduced food consumption during premating days 0-7 (-10%) and 0-13 (-8%)

- Statistically significantly decreased body weight change during premating days 0-7 (-85% below control), days 0-13 (-49%) and during mating (without statistical significance, -24%).

F0 females:

- Statistically significantly reduced food consumption during premating days 0-7 (-8%) and PND 4-7 (-11%)

- Body weight loss and a decrease of body weight change (-5.8 g) without statistical significance during PND 0-4 (lactation period)

F1 PUPS / CLINICAL EXAMINATIONS/ GROSS FINDINGS

- Statistically significantly decreased pup body weight on PND 1-13 (about 10% below control on PND 13, both sexes combined)

- Statistically significantly decreased pup body weight change on PND 1-7 (up to 19% below control, both sexes combined) and PND 1-13 (11% below control, both sexes combined)

Test groups 2 and 1 (25 and 5 mg/kg bw/d)

F0 PARENTAL ANIMALS / CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

- No test substance-related adverse findings

F1 PUPS / CLINICAL EXAMINATIONS/ GROSS FINDINGS

- No test substance-related adverse findings

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2016-06-21 to 2016-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

- Principle of test: The test substance was administered to 4 animals/sex/ orally by gavage for a maximum of 15 days at doses of 0, 50, and 150 mg/kg bw. One day after the last administration the animals were assessed by gross pathology.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: WE20PA15
- Expiration date of the batch: 2017-10-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room teperature

OTHER SPECIFICS:
Liquid/colorless, clear

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines. This Wistar rat strain (Crl:WI(Han)) is selected because extensive historical control data is available for these rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: ♂ ~14 -15 weeks, ♀ ~ 13 weeks
- Housing: individually, in polycarbonate cages type III
- Diet: ad libitum, Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, drinking water
- Acclimation period: ~9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

in suspension with 0.5 % Carboxymethylcellulose with 5 mg/100 mL Tween80.

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.5, 1.5 g/100 mL
- Amount of vehicle: 10 mL/kg bw

DOSAGE PREPARATION:
For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5 % carboxymethylcellulose with 5 mg/100 mL Tween80 suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guarantee that the test substance concentrations in the vehicle remained stable.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

15 days

Frequency of treatment:

daily administration

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: request of the sponsor

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily clinical observations, before the administration as well as within 2 h and within 5 h after the administration. Abnormalities and changes were documented for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: on day 3, 7, 10, and 14

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption will be recorded for days 0-3, 3-7, 7-10, and 10-14.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption were recorded for days 0-3, 3-7, 7-10 and 10-14.

HAEMATOLOGY: Yes
- Parameters checked: Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), Platelets, differential blood count, Reticulocytes

CLINICAL CHEMISTRY: Yes
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Serum y-glutamyl transferase, Inorg. phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animal/organ weights: anesthetized animals, adrenal glands, kidneys, liver, spleen

HISTOPATHOLOGY: Yes
Organ / Tissue fixation: The following organs or tissues were fixed in 4 % buffered formaldehyde solution:
All gross lesions, adrenal glands, kidneys, liver, spleen, stomach

Statistics:

Means and standard deviations were calculated. Additionally, a DUNNETT-test (two-sided) on water consumption, food consumption, body weight and body weight change was performed. For clinical pathology and the weight of anesthetized animals and absolute and relative organ weights Kruskal-Wallis and Wilcoxon tests were performed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

After administration of 50 mg/kg bw/d test substance, all animals showed salivation.
After administration of 150 mg/kg bw/d test substance, 3 out of 4 animals showed salivation, 1 out of 4 showed fur discolored, 1 out of 4 showed convulsions tonic-clonic, 1 out of 4 showed lateral position, 3 out of 4 showed hyperesthesia, and 1 out of 4 showed twitching.

Mortality:

mortality observed, treatment-related

Description (incidence):

After application of 150 mg/kg bw/d test substance, all test animals died within one day. No animal died at 50 mg/kg bw/d.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of surviving animals showed no differences compared to untreated control animals.

Food consumption and compound intake (if feeding study):

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Male treated animals showed an increased water intake.
Female treated animals showed a reduced water intake in the first week (d0 - d7) of administration.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch code 1, guideline conform study under GLP.

Additional information

The test substance was assessed in a combined repeated dose toxicity study with the reproductive and developmental screening test according to OECD Guideline 422 (BASF SE, 2018). The test substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 5, 25 and 75 mg/kg bodyweight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5 % Sodium carboxymethyl cellulose with 5 mg/100 mL Tween 80 suspension in drinking water). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, one day post-mating in males, and the entire gestation period as well as up to 22 days of the lactation period in females. Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions.

Regarding clinical examinations, transient salivation for a few minutes immediately after treatment was observed in mid- and high-dose female and male parental animals and was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. No adverse clinical signs were observed in any of the test groups.

Food consumption and body weight (change) were affected in F0 females and males of the highest test group (75 mg/kg bw/d). Food consumption of males was statistically significantly below the control during premating days 0-7 (around -10 %) and days 0-13 (around -8 %). Food consumption of females was also statistically significantly reduced during premating days 0-7 (around -8 % below control) and PND 4-7 (around -11 %).

Complementary, body weight (change) in high-dose F0 males was statistically significantly decreased during premating days 0-7 (-85 % below control), days 0-13 (-49 %) and during mating (without statistical significance, -24 %). High-dose females showed a body weight loss during PND 0-4 (decreased body weight change of 5.8 g). It occurred only in one study period during lactation and females gained weight from PND 4 onwards. However, due to the body weight loss, it was assessed as treatment-related and adverse.

Overall, the above-mentioned reduction in food consumption and decrease in body weight (change) of high-dose F0 males and females during different study periods were assessed as treatment-related and adverse. Test groups 1 and 2 showed no adverse effects in any of those parameters. Regarding clinical pathology no treatment-related, adverse effects were observed up to a dose of 75 mg/kg bw/d. Regarding pathology, the relative liver weight of males in test group 3 (75 mg/kg bw/d) was significantly increased (+10 %) and slightly above the historical control range. Histopathology of the liver in this test group revealed a minimal to slight centrilobular vacuolation of macrovesicular type in 5 out of 10 males, which was consistent with a fatty change (positive in the Oil-Red-O stain). Furthermore, 3 out of 10 males showed a minimal centrilobular hypertrophy. Since all of these changes were of low severity and were not associated with clinical chemical hepatic alterations, they were regarded as treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of the test substance was the mid dose of 25 mg/kg bw/d. At the highest tested dose, treatment-related adverse effects were observed including reduction in food consumption and a decrease in body weight (change) of male and female F0 parental animals.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.