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REACH

7,7-dimethyl-3-oxa-6-azaoctan-1-ol

EC number: 400-390-6 | CAS number: 87787-67-5 FLEXSORB-SE

Life Cycle description
Uses advised against

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:: screening for reproductive / developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: From 05 Jan 2021 to 28 Apr 2022
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 Jan 2021 to 28 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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:

29 July 2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

Specific details on test material used for the study:

TEST MATERIAL
- Batch identification: 0022239490
- Purity: 95.9%
- Physical state/appearance: liquid / yellow, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperature)
- Stability under storage condition was guaranteed
- Homogeneity: given
- Expiry date: 20 Jan 2022

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld
- Females: nulliparous and non-pregnant
- Age at study initiation: males: about 14-15 weeks old, females: about 13 weeks old
- Weight at study initiation: males: 388.5 (mean), females: 220.2 (mean)
- Housing of male and female animals: polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 (610 x 435 x 215 mm), up to 5 animals per sex and cage during pretreatment and up to 2 animals per sex and cage during premating
- Housing during mating, gestation, lactation and females after weaning: polycarbonate cages type III up to 1 animal per sex and cage with exception during mating (1 male/1 female per cage) and rearing up to post-natal day (PND) 13 (1 dam with her litter), pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation
- Housing during motor activity measurements: individually in polycarbonate cages type III, with wire covers (floor area of about 800 cm²) and small amounts of bedding material
- Additional information on housing: dust-free wooden bedding was used in this study, wooden gnawing blocks and large play tunnels were used for environmental enrichment, the cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured
- Diet: ad libitum, mouse and rat maintenance diet "GLP" (Garanovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum, supplied from water bottles
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Jan 2021 To: 30 Mar 2021

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of test substance preparation: in intervals, which took into account the analytical results of the stability verification (at least 7 days)
- Homogenization of test substance preparation achieved by mixing with a magnetic stirrer
- Storage of test substance preparation at room temperature

VEHICLE
- Concentration in vehicle: 0 g/100 mL, 0.25 g/100 mL, 0.75 g/100 mL, 2.50 g/100 mL
- Amount of vehicle: 10 mL/kg bw/day, the calculation of the administered volume was generally based on the most recent individual body weights

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during pre-mating) and twice during lactation of the study, 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses, respectively. At the same time, these samples were used as a concentration control. At the same time points, one additional sample from the mid concentration was taken for concentration control analysis.

The analytical investigations of the test substance preparations via GC confirmed that the stability of the test substance in the preparations over a period of at least 7 days at room temperature was given. As the mixtures were not stored longer than this time period, the stability was guaranteed. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in deionized water. All measured values for the test substance were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations.

Duration of treatment / exposure:

After the acclimatization period, the duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), as well as the entire gestation and lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. In detail, the male and female animals were sacrificed on day 38 and 63 after the beginning of substance administration, respectively.

Frequency of treatment:

The test substance or vehicle only was administered orally via gavage to the P0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor).

Dose / conc.:

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

Remarks:

Group 0
Control

Dose / conc.:

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

Remarks:

Group 1
Low-dose level

Dose / conc.:

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

Remarks:

Group 2
Mid-dose level

Dose / conc.:

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

Remarks:

Group 3
High-dose level

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose range finding study:
Two 14-day repeated dose toxicity studies were performed as range-finding studies investigating 15, 50, 150 and 500 mg/kg bw/d. Up to 150 mg/kg bw/d no test substance-related, relevant effects were observed. At 500 mg/kg severe signs of systemic toxicity were observed in both sexes. Furthermore, one female animal was found dead on study day 4 and one female was sacrificed moribund on study day 10. Based on the severe clinical findings, like piloerection, poor general condition, respirations, sounds/labored, and semi-closed eyelid, the remaining females of the 500 mg/kg bw/d test group were sacrificed ahead of schedule on study day 13.

- Animal assignment:
Only animals with regular estrous cycle were selected for randomization before the start of the treatment period. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled by a computer.

- Fasting period before blood sampling for clinical biochemistry: 16 to 20 hours

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: morbidity, pertinent behavioral changes, signs of overt toxicity
- All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period, starting in the morning
- Findings were ranked according to the degree of severity
- For observation, animals were removed from their cages and placed in a standard arena
- Parameters assessed: abnormal behavior in ""handling", fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
- Exceptions: (a) during the mating period all females with negative evidence of sperm were weighed on mating days 1, 7 and 14, (b) during the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20, (c) females with litter were weighed on the day after parturition (PND 0), 4, 7, 10 and 13, (d) females without litter and after weaning (PND 13) were weighed once a week
- Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly, these body weight data were soley used for the calculation of the dose volume

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was determined once a week for male and female parental animals, with the following exceptions: (a) food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female P0 animals), (b) food consumption of the P0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20, (c) food consumption of P0 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

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning of the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16-20 hours prior blood sampling
- How many animals: first 5 surviving parental males per group at termination and first 5- females with litters (in order of delivery) per group at PND 14
- Parameters (see table No. 1) were determined in blood with EDTA-K3 as anticoagulant using a particle counter
- In a clotting test prothrombin time (HQT, Hepatoquick’s test) was measured using a ball coagulometer

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning of the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16-20 hours prior blood sampling
- How many animals: first 5 surviving parental males per group at termination and first 5- females with litters (in order of delivery) per group at PND 14
- Parameters checked in table No. 2 were examined

SERUM HORMONES: Yes
- Time of blood sample collection: prior sacrifice
- Animals fasted: Yes (adults only)
- How many animals: from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13, from all dams at PND 14 and all males at termination
- T4 (total thyroxine) and TSH (thyroid stimulating hormone) was measured in blood samples from the adult males and the PND 13 pups, but not in samples from surplus PND 4 pups and dams at PND 14 (stored for 1 year after finalization of report)
- T3 was not measured

URINALYSIS: No

IMMUNOLOGY: No

NEUROBEHAVIOURAL EXAMINATION: Yes (functional observation battery)
- In the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period
- 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/stereotyp, 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), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test
- Motor activity measurements

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- The following weights were determined on all animals on schedule: anesthetized animals (final body weight), epididymides, ovaries, prostate (ventral and dorsolateral part together, fixed), seminal vesicles with coagulating glands (fixed), testes, thyroid glands (with parathyroid glands) (fixed), uterus with cervix
- The following weights were determined in 5 animals per sex/group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands (fixed), brain, heart, kidneys, liver, spleen, thymus (fixed) (all paired organs were weighed together (left and right))
- The following organs and tissues of all parental animals were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution (see table No. 3)

HISTOPATHOLOGY: Yes
- After fixation, the following organs were processed histotechnically, examined by light microscopy and assessment of findings recorded (see table No. 4)

Other examinations:

- Estrous cycle determinations
- Male reproduction data: mating index, fertility index
- Female reproduction and delivery data: mating index, fertility index, gestation index, live birth index, postimplantation loss
- Litter data: number and status at delivery, viability/mortality (viability index, survival index), sex ratio, body weight, anogenital distance, anogenital index, nipple/areola, necropsy, thyroid hormones

Statistics:

- Statistics of the clinical examinations see table No. 5
- Statistics of clinical pathology see table No. 6
- Statistics of pathology see table No. 7

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance were detected in any of the male and female P0 parental animals in any of the groups during the entire study period.
In the high-dose group (250 mg/kg bw/d) one male, on study day 34 only, and one female, on study day 3 only, showed respiration sounds. These isolated findings on a single day in two animals only were assessed as spontaneous in nature and not related to the test substance. Furthermore, blood in bedding and no litter were observed in one high-dose female. This single and isolated finding was assessed as spontaneous in nature and not related to treatment.
In the mid-dose group (75 mg/kg bw/d) a protruding eyeball, right, was observed in one male on study days 27-38 as well as in one female, left, on gestation days 9 -22 and lactation days 0-23. Without a dose-dependency these isolated findings in two animals only were assessed as spontaneous in nature and not related to treatment.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test-substance related, relevant findings were observed in the body weight of male and female animals in any phase of the study.
The decreased mean body weight changes of the low-dose parental females during lactation days 0-4 (-68.6%) and of the mid-dose parental females during lactation days 10-13 (-400.4%) were considered as spontaneous in nature and not related to treatment, because of missing dose dependency.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test-substance related, relevant finding was observed in food consumption of male and females animals in any phase of the study.
The decreased food consumption of the mid-dose P0 males at the beginning of the administration period (study day 0 to 7; -8.5%) and of the mid-dose females during lactation days 7-10 (-16.7%) were considered as spontaneous in nature and not related to treatment, because of missing dose-dependency.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.
At the end of the administration period, in males of test group 3 (250 mg/kg bw/d) platelet counts were significantly increased. The mean value was above, the median value at the upper border of the historical control range. Prothrombin time (HQT) was not changed. Therefore, this change was regarded as non-adverse if at all treatment related. Additionally, in males of test group 1 (25 mg/kg bw/d) absolute lymphocyte counts were significantly increased, but the change was not dose dependent and therefore, the alteration was regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test group 3 (250 mg/kg bw/d) total bile acids were (not statistically significantly) increased, but the values were within the historical control range. Additionally, in males of test groups 1 and 3 (25 and 250 mg/kg bw/d) potassium values were significantly increased. However, both means were within the historical control range. In males of test group 2 (75 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly decreased but the change was not dose dependent. Therefore, the mentioned alterations were regarded as incidental and not treatment related.
At lactational day 14, in dams of test group 3 (250 mg/kg bw/d), triglyceride levels were (not statistically significantly) increased. The values were slightly above the historical control range. However, this is the only altered clinical pathology parameter among these individuals. Therefore, this change was regarded as maybe treatment related, but non adverse.

Endocrine findings:

no effects observed

Description (incidence and severity):

In parental males and in male and female pups at PND13 (test groups 1, 2 and 3; 25, 75 and 250 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home cage observations: One male animal (250 mg/kg bw/d) showed respiration sounds. This single and isolated finding was assessed as spontaneous in nature and not related to treatment.

Motor activity measurements: Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group.
Comparing the single intervals with the control groups, significantly decreased values were measured for male animals of test group 2 (75 mg/kg bw/d) and test group 3 (250 mg/kg bw/d) at interval 4 and for male animals of test group 1 (25 mg/kg bw/d) and test group 3 (250 mg/kg bw/d) at intervals 12. These differences were regarded to be incidental and not related to treatment as these altered intervals occurred without relation to dose and the overall motor activity was not affected.
Comparing the single intervals with the control groups, significantly decreased values were measured for female animals of test group 1 (25 mg/kg bw/d) at interval 6 and for female animals of test group 1 (25 mg/kg bw/d), 2 (75 mg/kg bw/d, and 3 (250 mg/kg bw/d) at interval 7. These differences were regarded to be incidental and not related to treatment as these altered intervals occurred without relation to dose and the overall motor activity was not affected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to control group 0 (set to 100%), the mean absolute weights of kidneys and prostate were significantly decreased in males (see table No. 8). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
The significant decreases of the absolute weight of the kidneys in males of all test groups were within the historical control range and without a dose-dependent relationship. Therefore, they were regarded as incidental and not treatment-related. The significant absolute weight decreases of the prostate in males of test groups 1 and 3 were also within historical control values and were considered incidental and not related to treatment.

When compared to control group 0 (set to 100%), the mean relative weight of kidneys was significantly increased in females (see table No. 9).
The significant relative weight increase of the kidneys in females of test group 3 was within the historical control range and considered incidental and not treatment-related. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the kidneys of males with incidences and grading according to the table No. 10.
Compared to the control group, a treatment-related and dose-dependent increase of eosinophilic droplets in tubular epithelial cells was observed in test groups 2 and 3. The immunohistochemical staining pattern shows that the presence of alpha2u-globulin (nephropathy) is likely.
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 high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
Fertility was not affected.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Gestation index: The gestation index was 100% in test group 0 (control), test group 1 (25 mg/kg bw/d), test group 2 (75 mg/kg bw/d) and 90 % in test group 3 (250 mg/kg bw/d). This minor deviation from the current control value was within the range of the historical control data. Therefore, it was not assessed as treatment-related.

Postimplantation loss: The postimplantation loss was 7.7% in test group 0 (control), 8.5% in test group 1 (25 mg/kg bw/d), 9.0% in test group 2 (75 mg/kg bw/d) and 14.0% in test group 3 (250 mg/kg bw/d). All incidences were within the historical control data.

Pup mortality: In test group 3 (250 mg/kg bw/d) three pups were cannibalized and two pups were found dead. In test group 2 (75 mg/kg bw/d) five pups were stillborn, two pups were found dead and one pup was missing. In test group 1 (25 mg/kg bw/d) one pup was found dead. In test group 0 (control) one pup was cannibalized. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Pub weight: One male runt was found in litter of test group 0 (control). One female runt was found in litter of test group 1 (25 mg/kg bw/d), four female runts were found in one litter of test group 2 (75 mg/kg bw/d) and two were found in two litters of test group 3 (25 mg/kg bw/d). A relation to dosing was not observed, test substance-related effects did not occur.

Anogenital index of pups: The anogenital index of female pups in test group 2 (75 mg/kg bw/d: 0.83) was significantly increased but still within the range of historical control data (0.69 – 0.87).

Pup necropsy observation: One female pup of test group 1 (25 mg/kg bw/d) showed findings dilated renal pelvis and dilated ureter at gross necropsy. This finding occurred without any relation to dosing. Thus, it was not considered to be associated to the treatment.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

Tab. 8: Significantly altered absolute organ weights in males.

	Male animals
Test group (mg/kg bw/d)	1 (25)	2 (75)	3 (250)
Kidneys	90%*	85%*	92%*
Prostate	88%*	89%	86%*

*p ≤ 0.05; **p ≤ 0.01

Tab. 9: Significantly altered relative organ weight in females.

Female animals

Test group (mg/kg bw/d)

(25)

(75)

(250)

Kidneys

100%

101%

112%*

*p ≤ 0.05; **p ≤ 0.01

Tab. 10: Treatment-related histopathological findings in the kidneys of males.

	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (25)	2 (75)	3 (250)
No. of animals	10	10	10	10
Eosinophilic droplets	1	2	5	9
· Grade 1	1	2	4	1
· Grade 2			1	5
· Grade 3				3

Conclusions:

The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity was set to 250 mg/kg bw/day.

Executive summary:

Under the conditions of the OECD TG 422 and in compliance with GLP, a combined repeated dose toxicity study with the reproductive/developmental screening test was conducted in male and female Wistar rats. The test substance was administered daily as a solution to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 25 mg/kg bw/d (test group 1), 75 mg/kg bw/d (test group 2) and 250 mg/kg bw/d (test group 3). Deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Regarding clinical examinations, no test substance-related, adverse findings were observed up to a dose level of 250 mg/kg bw/d.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 250 mg/kg bw/d.

Regarding pathology, neither the organ weights nor the gross pathology showed treatment-related changes in males and females.

Histopathology revealed a treatment-related and dose-dependent increase of eosinophilic droplets in the tubular epithelium of the kidneys in males of test groups 2 (75 mg/kg bw/d) and 3 (250 mg/kg bw/d) (see table No. 10). An immunohistochemical stain performed exemplarily in two males of test group 3 revealed most likely the presence of an alpha2u-globulin nephropathy. This finding was not considered adverse, since no signs of cytotoxicity were observed in the tubular epithelium. Alpha2u-globulin is a male and rat-specific poor soluble protein synthesized in the male rat liver, filtered by the glomerulus and reabsorbed in the S2 segment of the proximal tubules where it is slowly hydrolyzed by lysosomal digestion. Reversible binding of the test substance or their metabolites to alpha2u-globulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that can be visible in form of characteristic eosinophilic droplets. Alpha2u-globulin nephropathy does not represent a risk for humans since they do not synthesize this protein.

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.

Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 250 mg/kg bw/day for male and female Wistar rats.

Cross-reference 2

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 05 Jan 2021 to 28 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

Specific details on test material used for the study:

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water

Details on exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details on mating procedure:

- M/F ratio per cage: 1:1, each female animal was paired with a predetermined male animal from the same dose group, females were placed in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning (deviations from the specified times were possible on weekends and public holidays)
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged in a polycarbonate cages type III (1 animal per cage)

Duration of treatment / exposure:

Frequency of treatment:

Dose / conc.:

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

Remarks:

Group 0
Control

Dose / conc.:

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

Remarks:

Group 1
Low-dose level

Dose / conc.:

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

Remarks:

Group 2
Mid-dose level

Dose / conc.:

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

Remarks:

Group 3
High-dose level

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: morbidity, pertinent behavioral changes, signs of overt toxicity
- All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period, starting in the morning
- Findings were ranked according to the degree of severity
- For observation, animals were removed from their cages and placed in a standard arena
- Parameters assessed: abnormal behavior in ""handling", fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
- Exceptions: (a) during the mating period all females with negative evidence of sperm were weighed on mating days 1, 7 and 14, (b) during the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20, (c) females with litter were weighed on the day after parturition (PND 0), 4, 7, 10 and 13, (d) females without litter and after weaning (PND 13) were weighed once a week
- Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly, these body weight data were soley used for the calculation of the dose volume

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was determined once a week for male and female parental animals, with the following exceptions: (a) food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female P0 animals), (b) food consumption of the P0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20, (c) food consumption of P0 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

NEUROBEHAVIOURAL EXAMINATION: Yes (functional observation battery)
- In the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period
- 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/stereotyp, 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), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test
- Motor activity measurements

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice of maternal animals on day 63 and of male animals on day 38
- The following weights were determined on all animals on schedule: anesthetized animals (final body weight), epididymides, ovaries, prostate (ventral and dorsolateral part together, fixed), seminal vesicles with coagulating glands (fixed), testes, thyroid glands (with parathyroid glands) (fixed), uterus with cervix
- The following weights were determined in 5 animals per sex/group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands (fixed), brain, heart, kidneys, liver, spleen, thymus (fixed) (all paired organs were weighed together (left and right))
- Organs examined (all animals): see table No. 1 (organs and tissues fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution)
- After fixation, the following organs were processed histotechnically, examined by light microscopy and assessment of findings recorded (see table No. 2)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Uterus with cervix
- Ovaries

Blood sampling:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning of the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16-20 hours prior blood sampling
- How many animals: first 5 surviving parental males per group at termination and first 5- females with litters (in order of delivery) per group at PND 14
- Parameters (see table No. 3) were determined in blood with EDTA-K3 as anticoagulant using a particle counter
- In a clotting test prothrombin time (HQT, Hepatoquick’s test) was measured using a ball coagulometer

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning of the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16-20 hours prior blood sampling
- How many animals: first 5 surviving parental males per group at termination and first 5- females with litters (in order of delivery) per group at PND 14
- Parameters checked in table No. 4 were examined

SERUM HORMONES: Yes
- Time of blood sample collection: prior sacrifice
- Animals fasted: Yes (adults only)
- How many animals: from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13, from all dams at PND 14 and all males at termination
- T4 (total thyroxine) and TSH (thyroid stimulating hormone) was measured in blood samples from the adult males and the PND 13 pups, but not in samples from surplus PND 4 pups and dams at PND 14 (stored for 1 year after finalization of report)
- T3 was not measured

Fetal examinations:

- Anogenital distance of all live rodent pups: Yes
- AGD was determined in all live male and female pups on PND 1 in randomized order, using a measuring ocular and AGD index calculated accordingly:
Anogenital index = anogenital distance [mm]/cubic root of pup weight [g]

Statistics:

- Statistics of the clinical examinations see table No. 5
- Statistics of clinical pathology see table No. 6
- Statistics of pathology see table No. 7

Indices:

MALE REPRODUCTION DATA:
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) X 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) X 100
* defined by a female with implants in the utero

FEMALE REPRODUCTION AND DELIVERY DATA:
Female mating index (%) = (number of females mated*/number of females placed with males) X 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant*/number of females mated**) X 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) X 100
* defined as the number of females with implants in utero

Live birth index (%) = (number of live born pups at birth/total number of pups born) X 100

Postimplantation loss (%) = (number of implantations - number of pups delivered/ number of implantations) X 100

OFFSPRING VIABILITY INDICES:
Viability index (%) = (number of live pups on day 4* after birth/number of live pups on the day of birth) X 100
* before standardization of litters (i.e. before culling)

Survival index (%) = (number of live pups on day 13 after birth/number of live pups on day 4* after birth) X 100
* after standardization of litters (i.e. after culling)

Historical control data:

Historical control data available

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Endocrine findings:

no effects observed

Description (incidence and severity):

In parental males and in male and female pups at PND13 (test groups 1, 2 and 3; 25, 75 and 250 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The postimplantation loss was 7.7% in test group 0 (control), 8.5% in test group 1 (25 mg/kg bw/d), 9.0% in test group 2 (75 mg/kg bw/d) and 14.0% in test group 3 (250 mg/kg bw/d). All incidences were within the historical control data.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was 22.4 days in test group 0 (control), test group 2 (75 mg/kg bw/d) and 3 (250 mg/kg bw/d). The mean duration of gestation was 22.2 days in test group 1 (25 mg/kg bw/d).

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All sperm positive females of test groups 0 to 3 (control; 25, 75 and 250 mg/kg bw/d) delivered pups, except for one female of test group 3 which did not deliver pups. Since this animal had implantation sites, the female fertility index was 100% in test groups 0, 1 2, and 3.
The gestation index was 100% in test group 0 (control), test group 1 (25 mg/kg bw/d), test group 2 (75 mg/kg bw/d) and 90 % in test group 3 (250 mg/kg bw/d). This minor deviation from the current control value was within the range of the historical control data. Therefore, it was not assessed as treatment-related.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

maternal systemic toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
Mean pup body weights and mean pup body weight change values of pups in test groups 1, 2 and 3 (25, 75 and 250 mg/kg bw/d) were comparable to the control group.
One male runt was found in litter of test group 0 (control). One female runt was found in litter of test group 1 (25 mg/kg bw/d), four female runts were found in one litter of test group 2 (75 mg/kg bw/d) and two were found in two litters of test group 3 (25 mg/kg bw/d). A relation to dosing was not observed, test substance-related effects did not occur.

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup survival during lactation (PND 0 – 4) was 99.3% in test group 0 (control), 100% in test group 1 (25 mg/kg bw/d), 95.7% in test group 2 (75 mg/kg bw/d) and 96.8% in test group 3 (250 mg/kg bw/d).
The survival index indicating pup survival during lactation (PND 4 – 13) was 100% in test groups 0 and 2 (control and 75 mg/kg bw/d), 98.8% in test groups 1 (25 mg/kg bw/d) and 98.6% in test group 3 (250 mg/kg bw/d).
Concerning mortality, in test group 3 (250 mg/kg bw/d) three pups were cannibalized and two pups were found dead. In test group 2 (75 mg/kg bw/d) five pups were stillborn, two pups were found dead and one pup was missing. In test group 1 (25 mg/kg bw/d) one pup was found dead. In test group 0 (control) one pup was cannibalized.
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 1 (25 mg/kg bw/d), one pup showed short tail. No clinical adverse findings were observed in pups of test groups 0, 2 and 3 (control, 75 and 250 mg/kg bw/d).

Skeletal malformations:

not examined

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One female pup of test group 1 (25 mg/kg bw/d) showed findings of dilated renal pelvis and dilated ureter at gross necropsy. This finding occurred without any relation to dosing. Thus, it was not considered to be associated to the treatment.
All other F1 pups of any test groups (0-3) did not show adverse findings during necropsy.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distance of all test substance treated male and female pups was comparable to the concurrent control values. The anogenital indices of all male and female pups in test group 0 (control), test group 1 (25 mg/kg bw/d), and test group 3 (250 mg/kg bw/d) as well as male pups of test group 2 (75 mg/kg bw/d) were comparable to the concurrent control values.
The anogenital index of female pups in test group 2 (75 mg/kg bw/d) was significantly increased but still within the range of historical control data.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

Tab. 8: Significantly altered absolute organ weights in males.

	Male animals
Test group (mg/kg bw/d)	1 (25)	2 (75)	3 (250)
Kidneys	90%*	85%*	92%*
Prostate	88%*	89%	86%*

*p ≤ 0.05; **p ≤ 0.01

Tab. 9: Significantly altered relative organ weight in females.

Female animals

Test group (mg/kg bw/d)

(25)

(75)

(250)

Kidneys

100%

101%

112%*

*p ≤ 0.05; **p ≤ 0.01

Tab. 10: Treatment-related histopathological findings in the kidneys of males.

	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (25)	2 (75)	3 (250)
No. of animals	10	10	10	10
Eosinophilic droplets	1	2	5	9
· Grade 1	1	2	4	1
· Grade 2			1	5
· Grade 3				3

Conclusions:

Based on the existing results of this study, the NOAEL for maternal systemic toxicity and maternal fertility as well as the NOAEL for pup development was determined to be 250 mg/kg bw/day, based on the absence of adverse effects observed at the highest dose level.

Executive summary:

Regarding clinical examinations and clinical pathology, no test substance-related, adverse findings were observed up to a dose level of 250 mg/kg bw/d. Gross pathology and organ weights did not show treatment-related changes in parental males and females.

Histopathology revealed a treatment-related and dose-dependent increase of eosinophilic droplets in the tubular epithelium of the kidneys in parental males of test groups 2 (75 mg/kg bw/d) and 3 (250 mg/kg bw/d) (see table No. 10) but was identified to be non-adverse and does not represent a risk for humans.

Concerning fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Mating behavior, conception, implantation and parturition were not affected.

Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth remained unaffected by the test substance treatment and occured in a non-treatment related manner.

Therefore, the no observed adverse effect level (NOAEL) for general and systemic toxicity was determined to be 250 mg/kg bw/day, the NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/day and the NOAEL for developmental toxicity was set to 250 mg/kg bw/day for male and female Wistar rats, based on the absence of adverse effects observed at the highest dose level (250 mg/kg bw/d).

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2022
Report date:: 2022

Materials and methods

Test guidelineopen all close all

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:: 29 July 2016

Test guideline 2

Qualifier:: according to guideline
Guideline:: other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:: July 2000

GLP compliance:: yes (incl. QA statement)
Remarks:: Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz
Limit test:: no

Test material

Test material information

Constituent 1

Reference substance name:: 7,7-dimethyl-3-oxa-6-azaoctan-1-ol
EC Number:: 400-390-6
EC Name:: 7,7-dimethyl-3-oxa-6-azaoctan-1-ol
Cas Number:: 87787-67-5
Molecular formula:: Hill formula: C8 H19 N O2 CAS formula: C8 H19 N O2
IUPAC Name:: 2-[2-(tert-butylamino)ethoxy]ethan-1-ol

Specific details on test material used for the study:: TEST MATERIAL
- Batch identification: 0022239490
- Purity: 95.9%
- Physical state/appearance: liquid / yellow, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperature)
- Stability under storage condition was guaranteed
- Homogeneity: given
- Expiry date: 20 Jan 2022

Test animals

Species:: rat
Strain:: Wistar
Remarks:: Crl:WI(Han)
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld
- Females: nulliparous and non-pregnant
- Age at study initiation: males: about 14-15 weeks old, females: about 13 weeks old
- Weight at study initiation: males: 388.5 (mean), females: 220.2 (mean)
- Housing of male and female animals: polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 (610 x 435 x 215 mm), up to 5 animals per sex and cage during pretreatment and up to 2 animals per sex and cage during premating
- Housing during mating, gestation, lactation and females after weaning: polycarbonate cages type III up to 1 animal per sex and cage with exception during mating (1 male/1 female per cage) and rearing up to post-natal day (PND) 13 (1 dam with her litter), pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation
- Housing during motor activity measurements: individually in polycarbonate cages type III, with wire covers (floor area of about 800 cm²) and small amounts of bedding material
- Additional information on housing: dust-free wooden bedding was used in this study, wooden gnawing blocks and large play tunnels were used for environmental enrichment, the cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured
- Diet: ad libitum, mouse and rat maintenance diet "GLP" (Garanovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum, supplied from water bottles
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Jan 2021 To: 30 Mar 2021

Administration / exposure

Route of administration:: oral: gavage
Vehicle:: water
Remarks:: deionized water
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
- Rate of test substance preparation: in intervals, which took into account the analytical results of the stability verification (at least 7 days)
- Homogenization of test substance preparation achieved by mixing with a magnetic stirrer
- Storage of test substance preparation at room temperature

VEHICLE
- Concentration in vehicle: 0 g/100 mL, 0.25 g/100 mL, 0.75 g/100 mL, 2.50 g/100 mL
- Amount of vehicle: 10 mL/kg bw/day, the calculation of the administered volume was generally based on the most recent individual body weights
Details on mating procedure:: - M/F ratio per cage: 1:1, each female animal was paired with a predetermined male animal from the same dose group, females were placed in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning (deviations from the specified times were possible on weekends and public holidays)
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- After successful mating each pregnant female was caged in a polycarbonate cages type III (1 animal per cage)
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: At the beginning (during pre-mating) and twice during lactation of the study, 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses, respectively. At the same time, these samples were used as a concentration control. At the same time points, one additional sample from the mid concentration was taken for concentration control analysis.

The analytical investigations of the test substance preparations via GC confirmed that the stability of the test substance in the preparations over a period of at least 7 days at room temperature was given. As the mixtures were not stored longer than this time period, the stability was guaranteed. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in deionized water. All measured values for the test substance were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations.
Duration of treatment / exposure:: After the acclimatization period, the duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), as well as the entire gestation and lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. In detail, the male and female animals were sacrificed on day 38 and 63 after the beginning of substance administration, respectively.
Frequency of treatment:: The test substance or vehicle only was administered orally via gavage to the P0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor).
Details on study schedule:: - Age at mating of the mated animals (P0 generation) in the study: approx. 13-14 weeks (males) and 12 weeks (females) (approx. 11-12 weeks old (males) and 10 weeks old (females) at treatment start, followed by 14 days of exposure before mating procedures)

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 mg/kg bw/day (actual dose received)
Remarks:: Group 0
Control

Doses / concentrations 2

Dose / conc.:: 25 mg/kg bw/day (actual dose received)
Remarks:: Group 1
Low-dose level

Doses / concentrations 3

Dose / conc.:: 75 mg/kg bw/day (actual dose received)
Remarks:: Group 2
Mid-dose level

Doses / concentrations 4

Dose / conc.:: 250 mg/kg bw/day (actual dose received)
Remarks:: Group 3
High-dose level

No. of animals per sex per dose:: 10
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose range finding study:
Two 14-day repeated dose toxicity studies were performed as range-finding studies investigating 15, 50, 150 and 500 mg/kg bw/d. Up to 150 mg/kg bw/d no test substance-related, relevant effects were observed. At 500 mg/kg severe signs of systemic toxicity were observed in both sexes. Furthermore, one female animal was found dead on study day 4 and one female was sacrificed moribund on study day 10. Based on the severe clinical findings, like piloerection, poor general condition, respirations, sounds/labored, and semi-closed eyelid, the remaining females of the 500 mg/kg bw/d test group were sacrificed ahead of schedule on study day 13.

- Animal assignment:
Only animals with regular estrous cycle were selected for randomization before the start of the treatment period. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled by a computer.

- Fasting period before blood sampling for clinical biochemistry: 16 to 20 hours

Examinations

Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: morbidity, pertinent behavioral changes, signs of overt toxicity
- All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period, starting in the morning
- Findings were ranked according to the degree of severity
- For observation, animals were removed from their cages and placed in a standard arena
- Parameters assessed: abnormal behavior in ""handling", fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
- Exceptions: (a) during the mating period all females with negative evidence of sperm were weighed on mating days 1, 7 and 14, (b) during the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20, (c) females with litter were weighed on the day after parturition (PND 0), 4, 7, 10 and 13, (d) females without litter and after weaning (PND 13) were weighed once a week
- Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly, these body weight data were soley used for the calculation of the dose volume

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was determined once a week for male and female parental animals, with the following exceptions: (a) food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female P0 animals), (b) food consumption of the P0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20, (c) food consumption of P0 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

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning of the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16-20 hours prior blood sampling
- How many animals: first 5 surviving parental males per group at termination and first 5- females with litters (in order of delivery) per group at PND 14
- Parameters (see table No. 1) were determined in blood with EDTA-K3 as anticoagulant using a particle counter
- In a clotting test prothrombin time (HQT, Hepatoquick’s test) was measured using a ball coagulometer

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning of the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, 16-20 hours prior blood sampling
- How many animals: first 5 surviving parental males per group at termination and first 5- females with litters (in order of delivery) per group at PND 14
- Parameters checked in table No. 2 were examined

SERUM HORMONES: Yes
- Time of blood sample collection: prior sacrifice
- Animals fasted: Yes (adults only)
- How many animals: from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13, from all dams at PND 14 and all males at termination
- T4 (total thyroxine) and TSH (thyroid stimulating hormone) was measured in blood samples from the adult males and the PND 13 pups, but not in samples from surplus PND 4 pups and dams at PND 14 (stored for 1 year after finalization of report)
- T3 was not measured

URINALYSIS: No

IMMUNOLOGY: No

NEUROBEHAVIOURAL EXAMINATION: Yes (functional observation battery)
- In the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period
- 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/stereotyp, 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), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test
- Motor activity measurements
Oestrous cyclicity (parental animals):: - For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear
- Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female P0 rats
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were killed and discarded
- Standardisation of litters was not performed in litters with less than 8 pups

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
(a) Number and status at delivery
(b) Viability/mortality: a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as Sundays or public holidays, the number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined, the number of live pups was calculated on the day after birth (PND 0), and on lactation days 4, 7 and 13
(c) Sex ratio: on the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle, later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line, the sex of the pups was finally confirmed at necropsy

Sex ratio = (number of live male or female pups on day 0 and 13/number of live male and female pups on day 0 and 13) X 100

(d) Body weight: pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13, the individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters, “runts” (pups that weigh less than 75% of the mean weight of the respective control pups) were defined on the basis of the body weights on PND 1

(e) Anogenital distance: anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle, AGD was determined in all live male and female pups on PND 1 in randomized order, using a measuring ocular

(f) Anogenital index = anogenital distance [mm]/cubic root of pup weight [g]

(g) Nipple/areola: all surviving male pups were examined for the presence of nipple/areola anlagen on PND 13, the number of nipple/areola anlagen was counted

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: all surviving animals on day 38
- Maternal animals: all surviving animals on day 63

GROSS PATHOLOGY: Yes
- The following weights were determined on all animals on schedule: anesthetized animals (final body weight), epididymides, ovaries, prostate (ventral and dorsolateral part together, fixed), seminal vesicles with coagulating glands (fixed), testes, thyroid glands (with parathyroid glands) (fixed), uterus with cervix
- The following weights were determined in 5 animals per sex/group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands (fixed), brain, heart, kidneys, liver, spleen, thymus (fixed) (all paired organs were weighed together (left and right))
- The following organs and tissues of all parental animals were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution (see table No. 3)

HISTOPATHOLOGY: Yes
- After fixation, the following organs were processed histotechnically, examined by light microscopy and assessment of findings recorded (see table No. 4)
Postmortem examinations (offspring):: GROSS EXAMINATION OF DEAD PUPS:
- On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation
- Remaining pups will be sacrificed under isoflurane anesthesia with CO2
- Blood was sampled for determination of thyroid hormone concentrations
- After sacrifice, the pups and the organs were assessed macroscopically
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations
- Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were archived without further processing
- All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically
- All pups without notable findings or abnormalities were discarded after their macroscopic evaluation
- Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted
- The remaining pups were sacrificed under isoflurane anesthesia with CO2, after sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically
Statistics:: - Statistics of the clinical examinations see table No. 5
- Statistics of clinical pathology see table No. 6
- Statistics of pathology see table No. 7
Reproductive indices:: MALE REPRODUCTION DATA:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for P0 breeding pairs
- For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) X 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) X 100
* defined by a female with implants in the utero

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for P0 females
- For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = (number of females mated*/number of females placed with males) X 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of femalespregnant*/number of females mated**) X 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) X 100
* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) X 100

Postimplantation loss (%) = (number of implantations - number of pups delivered/ number of implantations) X 100
Offspring viability indices:: Viability index (%) = (number of live pups on day 4* after birth/number of live pups on the day of birth) X 100
* before standardization of litters (i.e. before culling)

Survival index (%) = (number of live pups on day 13 after birth/number of live pups on day 4* after birth) X 100
* after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: No clinical signs or changes of general behavior, which may be attributed to the test substance were detected in any of the male and female P0 parental animals in any of the groups during the entire study period.
In the high-dose group (250 mg/kg bw/d) one male, on study day 34 only, and one female, on study day 3 only, showed respiration sounds. These isolated findings on a single day in two animals only were assessed as spontaneous in nature and not related to the test substance. Furthermore, blood in bedding and no litter were observed in one high-dose female. This single and isolated finding was assessed as spontaneous in nature and not related to treatment.
In the mid-dose group (75 mg/kg bw/d) a protruding eyeball, right, was observed in one male on study days 27-38 as well as in one female, left, on gestation days 9 -22 and lactation days 0-23. Without a dose-dependency these isolated findings in two animals only were assessed as spontaneous in nature and not related to treatment.
Mortality:: no mortality observed
Description (incidence):: No animal died prematurely in the present study.
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: No test-substance related, relevant findings were observed in the body weight of male and female animals in any phase of the study.
The decreased mean body weight changes of the low-dose parental females during lactation days 0-4 (-68.6%) and of the mid-dose parental females during lactation days 10-13 (-400.4%) were considered as spontaneous in nature and not related to treatment, because of missing dose dependency.
Food consumption and compound intake (if feeding study):: effects observed, non-treatment-related
Description (incidence and severity):: No test-substance related, relevant finding was observed in food consumption of male and females animals in any phase of the study.
The decreased food consumption of the mid-dose P0 males at the beginning of the administration period (study day 0 to 7; -8.5%) and of the mid-dose females during lactation days 7-10 (-16.7%) were considered as spontaneous in nature and not related to treatment, because of missing dose-dependency.
Food efficiency:: not examined
Ophthalmological findings:: not examined
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: No treatment-related, adverse changes among hematological parameters were observed.
At the end of the administration period, in males of test group 3 (250 mg/kg bw/d) platelet counts were significantly increased. The mean value was above, the median value at the upper border of the historical control range. Prothrombin time (HQT) was not changed. Therefore, this change was regarded as non-adverse if at all treatment related. Additionally, in males of test group 1 (25 mg/kg bw/d) absolute lymphocyte counts were significantly increased, but the change was not dose dependent and therefore, the alteration was regarded as incidental and not treatment related.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test group 3 (250 mg/kg bw/d) total bile acids were (not statistically significantly) increased, but the values were within the historical control range. Additionally, in males of test groups 1 and 3 (25 and 250 mg/kg bw/d) potassium values were significantly increased. However, both means were within the historical control range. In males of test group 2 (75 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly decreased but the change was not dose dependent. Therefore, the mentioned alterations were regarded as incidental and not treatment related.
At lactational day 14, in dams of test group 3 (250 mg/kg bw/d), triglyceride levels were (not statistically significantly) increased. The values were slightly above the historical control range. However, this is the only altered clinical pathology parameter among these individuals. Therefore, this change was regarded as maybe treatment related, but non adverse.
Endocrine findings:: no effects observed
Description (incidence and severity):: In parental males and in male and female pups at PND13 (test groups 1, 2 and 3; 25, 75 and 250 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:: not examined
Behaviour (functional findings):: effects observed, non-treatment-related
Description (incidence and severity):: Home cage observations: One male animal (250 mg/kg bw/d) showed respiration sounds. This single and isolated finding was assessed as spontaneous in nature and not related to treatment.

Motor activity measurements: Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group.
Comparing the single intervals with the control groups, significantly decreased values were measured for male animals of test group 2 (75 mg/kg bw/d) and test group 3 (250 mg/kg bw/d) at interval 4 and for male animals of test group 1 (25 mg/kg bw/d) and test group 3 (250 mg/kg bw/d) at intervals 12. These differences were regarded to be incidental and not related to treatment as these altered intervals occurred without relation to dose and the overall motor activity was not affected.
Comparing the single intervals with the control groups, significantly decreased values were measured for female animals of test group 1 (25 mg/kg bw/d) at interval 6 and for female animals of test group 1 (25 mg/kg bw/d), 2 (75 mg/kg bw/d, and 3 (250 mg/kg bw/d) at interval 7. These differences were regarded to be incidental and not related to treatment as these altered intervals occurred without relation to dose and the overall motor activity was not affected.
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):: Treatment-related findings were observed in the kidneys of males with incidences and grading according to the table No. 10.
Compared to the control group, a treatment-related and dose-dependent increase of eosinophilic droplets in tubular epithelial cells was observed in test groups 2 and 3. The immunohistochemical staining pattern shows that the presence of alpha2u-globulin (nephropathy) is likely.
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 high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
Fertility was not affected.
Histopathological findings: neoplastic:: no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:: no effects observed
Description (incidence and severity):: Estrous cycle data revealed regular cycles in the rearing F1 female animals of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was between 3.8 and 4.0 days.
Reproductive function: sperm measures:: not examined
Reproductive performance:: effects observed, non-treatment-related
Description (incidence and severity):: Male reproduction data:
The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups.
Fertility was proven in all of the P0 parental males of test groups 0, 1, 2 and 3 (0, 25, 75 and 250 mg/kg bw/d) within the scheduled mating interval to produce F1 litter. Thus, the male fertility index was 100% in control and test substance-treated test groups 1 to 3.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 2.8 days for the test group 0 (control), 2.4 days for test group 1 (25 mg/kg bw/d) and test group 2 (75 mg/kg bw/d) and 2.9 days for test group 3 (250 mg/kg bw/d).
All sperm positive females of test groups 0 to 3 (control; 25, 75 and 250 mg/kg bw/d) delivered pups, except one animal of test group 3. Since this animal had implantation sites, the female fertility index was 100% in all test groups.
The mean duration of gestation was 22.4 days in test group 0 (control), test group 2 (75 mg/kg bw/d and 3 (250 mg/kg bw/d). The mean duration of gestation was 22.2 days in test group 1 25 mg/kg bw/d).
The gestation index was 100% in test group 0 (control), test group 1 (25 mg/kg bw/d), test group 2 (75 mg/kg bw/d) and 90 % in test group 3 (250 mg/kg bw/d). This minor deviation from the current control value was within the range of the historical control data.Therefore, it was not assessed as treatment-related.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account.
The postimplantation loss was 7.7% in test group 0 (control), 8.5% in test group 1 (25 mg/kg bw/d), 9.0% in test group 2 (75 mg/kg bw/d) and 14.0% in test group 3 (250 mg/kg bw/d). All incidences were within the range of historical data.
The mean number of F1 pups delivered per dam remained unaffected (11.9 / 13.1 / 10.9 and 12.9 pups/dam in test groups 0 - 3, respectively). All incidences were within the range of historical data.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 / 100 / 95.4 and 100% in test groups 0 - 3, respectively. The number of stillborn pups was not significantly different between the test groups.

None of the parameter assessed for male and female reproduction as well as delivery showed a treatment-related effect.

Effect levels (P0)

open all close all

1

Dose descriptor:: NOAEL
Remarks:: reproduction toxicity
Effect level:: >= 250 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable due to absence of adverse toxic effects

2

Dose descriptor:: NOAEL
Remarks:: systemic toxicity
Effect level:: >= 250 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:: no

Results: F1 generation

General toxicity (F1)

Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: In test group 1 (25 mg/kg bw/d), one pup showed short tail. No clinical adverse findings were observed in pups of test groups 0, 2 and 3 (control, 75 and 250 mg/kg bw/d).
Mortality / viability:: mortality observed, non-treatment-related
Description (incidence and severity):: The viability index indicating pup survival during lactation (PND 0 – 4) was 99.3% in test group 0 (control), 100% in test group 1 (25 mg/kg bw/d), 95.7% in test group 2 (75 mg/kg bw/d) and 96.8% in test group 3 (250 mg/kg bw/d).
The survival index indicating pup survival during lactation (PND 4 – 13) was 100% in test groups 0 and 2 (control and 75 mg/kg bw/d), 98.8% in test groups 1 (25 mg/kg bw/d) and 98.6% in test group 3 (250 mg/kg bw/d).
Concerning mortality, in test group 3 (250 mg/kg bw/d) three pups were cannibalized and two pups were found dead. In test group 2 (75 mg/kg bw/d) five pups were stillborn, two pups were found dead and one pup was missing. In test group 1 (25 mg/kg bw/d) one pup was found dead. In test group 0 (control) one pup was cannibalized.
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: Mean pup body weights and mean pup body weight change values of pups in test groups 1, 2 and 3 (25, 75 and 250 mg/kg bw/d) were comparable to the control group.
One male runt was found in litter of test group 0 (control). One female runt was found in litter of test group 1 (25 mg/kg bw/d; pup No. 116-11), four female runts were found in one litter of test group 2 (75 mg/kg bw/d) and two were found in two litters of test group 3 (25 mg/kg bw/d).
A relation to dosing was not observed, test substance-related effects did not occur.
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: no effects observed
Description (incidence and severity):: In male and female pups at PND13 (test groups 1, 2 and 3; 25, 75 and 250 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:: not examined
Sexual maturation:: not examined
Anogenital distance (AGD):: effects observed, non-treatment-related
Description (incidence and severity):: The anogenital distance of all test substance treated male and female pups was comparable to the concurrent control values.
The anogenital indices of all male and female pups in test group 0 (control), test group 1 (25 mg/kg bw/d), and test group 3 (250 mg/kg bw/d) as well as male pups of test group 2 (75 mg/kg bw/d) were comparable to the concurrent control values.
The anogenital index of female pups in test group 2 (75 mg/kg bw/d: 0.83) was significantly increased but still within the range of historical control data.
Nipple retention in male pups:: no effects observed
Description (incidence and severity):: No test substance related findings could be observed in test groups 1 to 3 (25, 75 and 250 mg/kg bw/d) when compared to the control group (test group 0).
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: One female pup of test group 1 (25 mg/kg bw/d) showed findings dilated renal pelvis and dilated ureter at gross necropsy.
This finding occurred without any relation to dosing. Thus, it was not considered to be associated to the treatment.
All other F1 pups of any test groups (0-3) did not show adverse findings during necropsy.
Histopathological findings:: not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):: not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:: not examined

Details on results (F1)

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Effect levels (F1)

Dose descriptor:: NOAEL
Remarks:: developmental toxicity
Generation:: F1
Effect level:: >= 250 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:: no

Overall reproductive toxicity

Reproductive effects observed:: no

Any other information on results incl. tables

Tab. 8: Significantly altered absolute organ weights in males.

	Male animals
Test group (mg/kg bw/d)	1 (25)	2 (75)	3 (250)
Kidneys	90%*	85%*	92%*
Prostate	88%*	89%	86%*

*p ≤ 0.05; **p ≤ 0.01

Tab. 9: Significantly altered relative organ weight in females.

Female animals

Test group (mg/kg bw/d)

(25)

(75)

(250)

Kidneys

100%

101%

112%*

*p ≤ 0.05; **p ≤ 0.01

Tab. 10: Treatment-related histopathological findings in the kidneys of males.

	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (25)	2 (75)	3 (250)
No. of animals	10	10	10	10
Eosinophilic droplets	1	2	5	9
· Grade 1	1	2	4	1
· Grade 2			1	5
· Grade 3				3

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of reproduction and developmental toxicity up to the highest tested dose level. Therefore, a NOAEL for reproductive performance and fertility as well as for developmental toxicity was set to 250 mg/kg bw/day.

Executive summary:

Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth remained unaffected by the test substance treatment.

Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 250 mg/kg bw/day, the NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/day and the NOAEL for developmental toxicity was set to 250 mg/kg bw/day for male and female Wistar rats.

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7,7-dimethyl-3-oxa-6-azaoctan-1-ol

Toxicity to reproduction

Administrative data

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Reference

Reference

Data source

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Materials and methods

Test guidelineopen allclose all

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Constituent 1

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Administration / exposure

Doses / concentrationsopen allclose all

Examinations

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Reproductive function / performance (P0)

Effect levels (P0)

Target system / organ toxicity (P0)

Results: F1 generation

General toxicity (F1)

Developmental neurotoxicity (F1)

Developmental immunotoxicity (F1)

Details on results (F1)

Effect levels (F1)

Target system / organ toxicity (F1)

Overall reproductive toxicity

Any other information on results incl. tables

Applicant's summary and conclusion

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Test guidelineopen all close all

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