Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline required

Principles of method if other than guideline:

14-day dose-range finding study with investigation of clinical chemistry, heamatology, body weight, food consumption, organ weight of liver, kidney, adrenal gland and spleen

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

4 animals per sex and group

Route of administration:

oral: gavage

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

Observations and examinations performed and frequency:

clinical chemistry, heamatology, body weight, food consumption

Sacrifice and pathology:

organ weight of liver, kidney, adrenal gland and spleen

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

male:
high dose group: Salivation (4/4), Respiration sounds (3/4), Plough nose-first into bedding (3/4), Abdominal position (3/4), Soft feces (4/4), Diarrhea (2/4), Piloerection (3/4), Fur smeared (3/4), Semniclosed eyelid (3/4), Closed eyelid (1/4)
mid dose group: Salivation (4/4), Respiration sounds (1/4)
low dose group: Salivation (1/4), Respiration sounds (1/4)

female:
high dose group: Salivation (4/4), Respiration sounds (3/4), Plough nose-first into bedding (4/4), Soft feces (1/4), Diarrhea (2/4), Piloerection (4/4), Fur smeared (1/4), Closed eyelid (1/4)
mid dose group: Salivation (3/4), Respiration sounds (3/4), Plough nose-first into bedding (2/4), Nose discharge (1/4), Piloerection (1/4), Semniclosed eyelid (1/4)
low dose group: Salivation (1/4), Respiration sounds (1/4)

Mortality:

mortality observed, treatment-related

Description (incidence):

female:
high dose group: 2/4

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

male:
high dose group: significantly decreased body weight gain

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

male:
high dose group: significantly decreased

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

minimal effects

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

minimal effects

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

males:
high dose group: Forestomach focus (4/4), Forestomach thickening of wall (2/4), Glandular stomach focus (2/4)
mid dose group: Esophagus discoloration (1/4), Forestomach thickening of wall (1/4)

female:
high dose group: Forestomach focus (1/4), Lungs discoloration (1/4)
mid dose group: Forestomach focus (1/4), Forestomach thickening of wall (1/4)

Neuropathological findings:

not examined

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

Critical effects observed:

no

Conclusions:

In range finding experiment (repeated dose toxicity study, 14 days) signs of systematic toxicity were observed at 300 mg/kg bw/d (i.a. respiration sounds, plough nose-first into bedding, and piloerection) and at 1000 mg/kg bw/d (i.a. mortality). The substance appeared to be irritating to the stomach of the mid and high dose group in the study. These findings are in accordance with the classification for skin and eye irritation based on the representative studies.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, 55116 Mainz, Germany

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate amount of test substance was weighed out depending on the desired concentration. Ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were prepared in such a manner that the stability was guaranteed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in ultra-pure water

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

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, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 11-12 weeks (male animals); about 10 weeks (female animals)
- Weight at study initiation: males: 368.3 g (mean); females: 215.2 g (mean)
The weight variation of the animals used did not exceed 20 % of the mean weight of each sex.
- Housing:
- during pretreatment: housed together (up to 5 animals per sex and cage)
- druing study period: individually

- Diet: ad libitum; ground Kliba maintenance diet mouse/rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- 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 was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE.

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

IN-LIFE DATES: From: 19 Sep 2017 To: 08 Nov 2017 (males); 05 Dec 2017 (females)

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least.
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with deionized water and mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.20, 0.75, 3.00 g/100 mL
- Amount of vehicle: 10 mL/kg bw/d
- Purity: deionized water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above-mentioned time points, additionally, one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample (described by the suffix “R”) was retained.
The samples collected at the beginning and during lactation period were analyzed in the Analytical Laboratory. Additionally, the reserve samples of the lowest concentration collected at the beginning of the study were analyzed. All further samples were not analyzed and stored frozen (at -20 °C) in the Laboratory of the Mechanistic Toxicology until the finalization of the study.

The reserve samples (sample no. 3R to 5R, mean 96%) of the low dose of premating period were measured due to slightly too high concentration in the primary samples (sample no. 3 to 5, mean 111%) considering the specification of the test facility (90-110%). No deviation was observed in the samples of the low dose (sample no. 26 to 28, mean 108%) collected towards the end of the study.

Thereby, all concentration analyses confirmed the nominal concentration in the test substance preparations. All analytical values were within the specification of the test facility: 90-110% of the nominal concentration.
Considering the three samples collected from one test substance preparation (samples no. 3-5, 3R-5R, 7-9, 26-28, or 30-32) only minor differences were observed within the three samples.

Therefore, the test item was considered as homogeneously distributed in the
vehicle (see Part III, Supplement).

Duration of treatment / exposure:

males: 29 days; females: 56 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

Once daily at approximately the same time in the morning
(Females in labor were not treated.)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high-dose group

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

low-dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the corresponding range finding experiment (repeated dose toxicity study, 14 days, 01R0407/16R119) signs of systematic toxicity were observed at 300 mg/kg bw/d (i.a. respiration sounds, plough nose-first into bedding, and piloerection) and at 1000 mg/kg bw/d (i.a. mortality). The substance appeared to be irritating to the stomach of the mid and high dose group in the study.These findings are in accordance with the classification for skin and eye irritation based on the representative studies.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration
- parturition and lactation behavior of the dams: in the mornings in combination with the daily clinical inspection
- on weekday afternoons (except Saturday, Sunday and public holidays): parturition behavior of the dams
- pups: examined daily for clinical symptoms (including gross-morphological findings)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration and at weekly intervals during administration
- Examined parameters: 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 for female parental animals:
• 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
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Pups: weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13
- individual pup weights: determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week for male and female parental animals
- exceptions:
• 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, 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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes, see table 1
- Time schedule for collection of blood: at termination, study day 29 (males); at PND 14, from study day 50 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.
- blood smears prerared and stained according to WRIGHT, not evaluated because of non-ambiguous results of the differential blood cell counts

CLINICAL CHEMISTRY: Yes, see table 2
- Time schedule for collection of blood: at termination, study day 29 (males); at PND 14, from study day 50 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- functional observational battery performed 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 (Male: study day 27; Female: study day 55) starting at about 10.00 h in a randomized sequence
- Parameters: passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests
- Ranking: according to the degree of severity, if applicable
- Examined observations:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• 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 (auditory 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, other findings

Motor activity assessment:
- Time schedule: at the end of the administration period rom 14:00 h onwards on the same day as the FOB was performed
- Animals: the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Method: TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany

Estrous cycle:
- Time schedule: daily analysis of vaginal smear for a minimum of 2 weeks prior to mating, continued throughout the pairing period until the female exhibited evidence of copulation, at necropsy
- Animals: all F0 female parental rats

Male reproduction data:
- Parameters: pairing partners, number of mating days until vaginal sperm in the female animals, gestational status of the females were recorded
- Animals: in all F0 breeding pairs

Female reproduction and delivery data
- Parameters: pairing partners, number of mating days until vaginal sperm, gestational status
Animals: in all F0 females

PUPS (thyroid hormones: see table 3)
- Sex ratio at day of birth by observing the distance between anus and the base of the genital tubercle
- Anogenital distance determined in all male and female pups on PND 1
- Nipple/areola anlagen presence observation on PND 13 of the lactation phase
- Necropsy observation on PND 4 of surplus pups and blood sampled for thyroid hormone concentration measurement
- Necropsy observation on PND 13 of one animal per litter and sex + blood sampling for thyroid measurement (stillborn pups and dead pups before PND13 observed the same way)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 4)

Statistics:

see table 5

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During pre-mating slight salivation after treatment was seen in one male and one female animal on day 4 and piloerection was seen in two males and two females of test group 3 (300 mg/kg bw/d) for up to 3 consequent days. During mating slight to moderate salivation after treatment was seen in two males of test group 3 up to 4 consequent days. During postmating moderate salivation after treatment was seen in two males on post-mating day 0. During gestation alopecia was observed in each one female animal of control group, test group 1 and 2 (20 and 75 mg/kg bw/d) and slight salivation after treatment was observed in two females of test group 3 (300 mg/kg bw/d) on up to 3 consequent days. During the lactation period alopecia still was seen in the same three animals of control group, test group 1 and 2. Furthermore, slight salivation after treatment was seen in three females of test group 3 on single days and one female of test group 3 showed respiration sounds on lactation day 0.
The salivation observed only after treatment at any time point the study were assessed as treatment-related but considered to be caused by bad taste or local irritation. Therefore, it was assessed as non-adverse.

A swelling of the mammary line was observed in three females of control group, four females of test group 1 and each in one female of test groups 2 and 3 on lactation day 14. This finding showed no significant difference to control and no dose-dependency. It was considered as incidental and likely to be a side effect of weaning the litter on PND 13.

Due to animals with delivery in the afternoon gaps were existing for gestation day 22 for two animals (animal nos. 120 and 140) and for lactation day 0 for eight animals (animal nos. 103, 108, 120, 121, 128, 132, 137, 140). These gaps are caused by the change of the phase (from gestation to lactation) within one day not by missing clinical observations.

All findings were considered to be treatment-related but not adverse.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- mean body weights of all male and female F0 generation parental animals in all test substance-treated groups: comparable to the concurrent control values
- exception: males of test group 3 (300 mg/kg bw/d) had significantly decreased body weight on pre-mating day 13 (-4.5%), not observed on studies days thereafter > assessed as treatment-related but not adverse
- bw changes significantly decreased: in male animals of test group 3 (-79.1%), test group 2 (75 mg/kg bw/d; -34.3%) during premating, not observed on studies days thereafter > assessed as treatment-related but not adverse
- changes seen in males of test group 1 (20 mg/kg bw/d; -34.2%) during premating, not observed on studies days thereafter > assessed as treatment-related but not adverse

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the F0 animals in all test groups was not influenced by the treatment throughout the entire study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Haematological findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia seen in one female animal each in test groups 2 and 1 as well as the control group was not considered to be test substance-related.

In males of test groups 1 (20 mg/kg bw/d), 2 (75 mg/kg bw/d) and 3 (300 mg/kg bw/d) number of rearings was reduced (-35%, -55% and -39%) and in females of test groups 1 and 3 number of rearings was increased (43% and 27%).
Since these findings were neither statistically significant nor dose-dependent altered, they were not considered to be treatment-related.

In female animals of test group 3 (300 mg/kg bw/d) a significantly decrease of beam interruptions was recorded for interval 8. However, since this finding was an isolated single event and no further significant changes were observed in the respective sum of all intervals, this finding was regarded incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- decrease in absolute testes weight of test group 2 males (75 mg/kg bw/d) as well as the changes in relative weights in kidneys, seminal vesicles and testes in different test groups were all regarded to be incidental and not related to treatment for the following reasons:
- there was no dose-response relationship, in test group 3 (300 mg/kg bw/d) no histopathologic findings were observed that could explain the weight increase and a slightly and not significantly reduced terminal body weight in test group 3 males (300 mg/kg bw/d) aggravated the relative increase in kidneys and testes weight
- please refer to table 6 and table 7

Gross pathological findings:

effects observed, non-treatment-related

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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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.

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance and anogenital index were significantly decreased in male pups of test group 2 (-5.29% and -4.43%, respectively). Furthermore, anogenital index was significantly reduced in males of test group 3 (-4.71%).
All determined anogenital distances and indices were still in the range of historical control data (anogenital distance: 2.91 – 3.36 and anogenital index: 1.52 – 1.75) and therefore regarded as incidental and not related to treatment.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Table 6: Mean absolute weights compared to control group with significantly change

	Male animals
Test group (mg/kg bw/d)	1 (20)	2 (75)	3 (300)
Testes	98%	95%*	105%

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group.

Table 7: Mean relative weights compared to control group with significantly change

	Male animals
Test group (mg/kg bw/d)	1 (20)	2 (75)	3 (300)
Kidneys	100%	102%	111%*
Seminal vesicle	123%**	110%	112%
Testes	100%	98%	110%*

*p <= 0.05; **p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The various analyses confirmed

·     no chemical and microbiological contaminatations in the diet in accordance with Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA),,

·     no analytical findings in drinking water in accordance with the German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990),

·     stability of the test substance in deionized water was demonstrated for a period of 7 days at room temperature,

·     the homogeneous distribution of the test substance in the vehicle,

·     the correctness of the prepared concentrations.

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no treatment-related adverse findings in parental animals.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest tested dose 300 mg/kg bw/d in both sexes of parental animals.

Executive summary:

The test substance was administered daily as liquid preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at nominal doses of 20, 75 and 300 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 (deionized water). The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 1 day post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females up to one day prior to the

day of schedule sacrifice of the animals.

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

Test group 3: 300 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

Test group 2: 75 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

Test group 1: 20 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no treatment-related adverse findings in parental animals.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest tested dose 300 mg/kg bw/d in both sexes of parental animals.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, 55116 Mainz, Germany

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate amount of test substance was weighed out depending on the desired concentration. Ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were prepared in such a manner that the stability was guaranteed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in ultra-pure water

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Sex: male/female
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 11-12 weeks (male animals); about 10 weeks (female animals)
- Weight at study initiation: males: 368.3 g (mean); females: 215.2 g (mean)
The weight variation of the animals used did not exceed 20 % of the mean weight of each sex.
- Housing:
- during pretreatment: housed together (up to 5 animals per sex and cage)
- druing study period: individually

- Diet: ad libitum; ground Kliba maintenance diet mouse/rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- 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 was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE.

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

IN-LIFE DATES: From: 19 Sep 2017 To: 08 Nov 2017 (males); 05 Dec 2017 (females)

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least.
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with deionized water and mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.20, 0.75, 3.00 g/100 mL
- Amount of vehicle: 10 mL/kg bw/d
- Purity: deionized water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above-mentioned time points, additionally, one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample (described by the suffix “R”) was retained.
The samples collected at the beginning and during lactation period were analyzed in the Analytical Laboratory. Additionally, the reserve samples of the lowest concentration collected at the beginning of the study were analyzed. All further samples were not analyzed and stored frozen (at -20 °C) in the Laboratory of the Mechanistic Toxicology until the finalization of the study.

The reserve samples (sample no. 3R to 5R, mean 96%) of the low dose of premating period were measured due to slightly too high concentration in the primary samples (sample no. 3 to 5, mean 111%) considering the specification of the test facility (90-110%). No deviation was observed in the samples of the low dose (sample no. 26 to 28, mean 108%) collected towards the end of the study.

Thereby, all concentration analyses confirmed the nominal concentration in the test substance preparations. All analytical values were within the specification of the test facility: 90-110% of the nominal concentration.
Considering the three samples collected from one test substance preparation (samples no. 3-5, 3R-5R, 7-9, 26-28, or 30-32) only minor differences were observed within the three samples.

Therefore, the test item was considered as homogeneously distributed in the
vehicle (see Part III, Supplement).

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as gestation day GD0
- Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
- After successful mating each pregnant female was caged individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop- Rauxel, Germany with nesting material (cellulose wadding) towards the end of gestation

Duration of treatment / exposure:

males: 29 days; females: 56 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

Once daily at approximately the same time in the morning
(Females in labor were not treated.)

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

low-dose group

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
In the corresponding range finding experiment (repeated dose toxicity study, 14 days, 01R0407/16R119) signs of systematic toxicity were observed at 300 mg/kg bw/d (i.a. respiration sounds, plough nose-first into bedding, and piloerection) and at 1000 mg/kg bw/d (i.a. mortality). The substance appeared to be irritating to the stomach of the mid and high dose group in the study.These findings are in accordance with the classification for skin and eye irritation based on the representative studies.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration
- parturition and lactation behavior of the dams: in the mornings in combination with the daily clinical inspection
- on weekday afternoons (except Saturday, Sunday and public holidays): parturition behavior of the dams
- pups: examined daily for clinical symptoms (including gross-morphological findings)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration and at weekly intervals during administration
- Examined parameters: 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 for female parental animals:
• 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
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Pups: weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13
- individual pup weights: determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week for male and female parental animals
- exceptions:
• 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, 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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes, see table 1
- Time schedule for collection of blood: at termination, study day 29 (males); at PND 14, from study day 50 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.
- blood smears prerared and stained according to WRIGHT, not evaluated because of non-ambiguous results of the differential blood cell counts

CLINICAL CHEMISTRY: Yes, see table 2
- Time schedule for collection of blood: at termination, study day 29 (males); at PND 14, from study day 50 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- functional observational battery performed 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 (Male: study day 27; Female: study day 55) starting at about 10.00 h in a randomized sequence
- Parameters: passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests
- Ranking: according to the degree of severity, if applicable
- Examined observations:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• 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 (auditory 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, other findings

Motor activity assessment:
- Time schedule: at the end of the administration period rom 14:00 h onwards on the same day as the FOB was performed
- Animals: the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Method: TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany

Ovaries and uterine content:

HAEMATOLOGY: Yes, see table 1

Estrous cycle:
- Time schedule: daily analysis of vaginal smear for a minimum of 2 weeks prior to mating, continued throughout the pairing period until the female exhibited evidence of copulation, at necropsy
- Animals: all F0 female parental rats

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups or 2 preferably pups/litter were killed and discarded.
- If individual litters did not have 4 pups/sex: litters were processed that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- check for dead or moribund pups: 2x/d on workdays (once in the morning and once in the afternoon)
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities, organs assessed macroscopically; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY, see table 3
- on PND 13, blood sampling for determination of thyroid hormone concentration
- thyroid glands/parathyroid glands fixed and pathology examinations

Statistics:

see table 5

Indices:

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 utero

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 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

Historical control data:

Available for: female matoing index, female fertility index, live birth indices, postimplantation loss, pup body weight data, anogenital distance/anogenital index of pups F1, clinical pathology F0

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During pre-mating slight salivation after treatment was seen in one male and one female animal on day 4 and piloerection was seen in two males and two females of test group 3 (300 mg/kg bw/d) for up to 3 consequent days. During mating slight to moderate salivation after treatment was seen in two males of test group 3 up to 4 consequent days. During postmating moderate salivation after treatment was seen in two males on post-mating day 0. During gestation alopecia was observed in each one female animal of control group, test group 1 and 2 (20 and 75 mg/kg bw/d) and slight salivation after treatment was observed in two females of test group 3 (300 mg/kg bw/d) on up to 3 consequent days. During the lactation period alopecia still was seen in the same three animals of control group, test group 1 and 2. Furthermore, slight salivation after treatment was seen in three females of test group 3 on single days and one female of test group 3 showed respiration sounds on lactation day 0.
The salivation observed only after treatment at any time point the study were assessed as treatment-related but considered to be caused by bad taste or local irritation. Therefore, it was assessed as non-adverse.

A swelling of the mammary line was observed in three females of control group, four females of test group 1 and each in one female of test groups 2 and 3 on lactation day 14. This finding showed no significant difference to control and no dose-dependency. It was considered as incidental and likely to be a side effect of weaning the litter on PND 13.

Due to animals with delivery in the afternoon gaps were existing for gestation day 22 for two animals (animal nos. 120 and 140) and for lactation day 0 for eight animals (animal nos. 103, 108, 120, 121, 128, 132, 137, 140). These gaps are caused by the change of the phase (from gestation to lactation) within one day not by missing clinical observations.

All findings were considered to be treatment-related but not adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- mean body weights of all male and female F0 generation parental animals in all test substance-treated groups: comparable to the concurrent control values
- exception: males of test group 3 (300 mg/kg bw/d) had significantly decreased body weight on pre-mating day 13 (-4.5%), not observed on studies days thereafter > assessed as treatment-related but not adverse
- bw changes significantly decreased: in male animals of test group 3 (-79.1%), test group 2 (75 mg/kg bw/d; -34.3%) during premating, not observed on studies days thereafter > assessed as treatment-related but not adverse
- changes seen in males of test group 1 (20 mg/kg bw/d; -34.2%) during premating, not observed on studies days thereafter > assessed as treatment-related but not adverse

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

Food consumption of the F0 animals in all test groups was not influenced by the treatment throughout the entire study period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Haematological findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia seen in one female animal each in test groups 2 and 1 as well as the control group was not considered to be test substance-related.

In males of test groups 1 (20 mg/kg bw/d), 2 (75 mg/kg bw/d) and 3 (300 mg/kg bw/d) number of rearings was reduced (-35%, -55% and -39%) and in females of test groups 1 and 3 number of rearings was increased (43% and 27%).
Since these findings were neither statistically significant nor dose-dependent altered, they were not considered to be treatment-related.

In female animals of test group 3 (300 mg/kg bw/d) a significantly decrease of beam interruptions was recorded for interval 8. However, since this finding was an isolated single event and no further significant changes were observed in the respective sum of all intervals, this finding was regarded incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- decrease in absolute testes weight of test group 2 males (75 mg/kg bw/d) as well as the changes in relative weights in kidneys, seminal vesicles and testes in different test groups were all regarded to be incidental and not related to treatment for the following reasons:
- there was no dose-response relationship, in test group 3 (300 mg/kg bw/d) no histopathologic findings were observed that could explain the weight increase and a slightly and not significantly reduced terminal body weight in test group 3 males (300 mg/kg bw/d) aggravated the relative increase in kidneys and testes weight

Gross pathological findings:

effects observed, non-treatment-related

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):

All observations were comparable to control animals.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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.

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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was 1 stillborn pup recorded in control group and test group 1, 4 stillborn pups recorded in test group 2 and 2 stillborn pups recorded in test group 3.
The postimplantation loss was 6.3% in test group 0, 3.3% in test group 1 (20 mg/kg bw/d), 2.4% in test group 2 (75 mg/kg bw/d) and 5.3% in test group 3 (300 mg/kg bw/d).
These findings 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 (0.0% - 18.1%) of the historical control data.

Total litter losses by resorption:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The rate live birth indices were 99.1% in test group 0, 99.2% in test group 1 (20 mg/kg bw/d), 96.8% in test group 2 (75 mg/kg bw/d) and 98.1% in test group 3 (300 mg/kg bw/d). The values of test group 1, 2 and 3 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.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation was similar in all test groups (22.2 days in test group 0; 22.0 days in test groups 1 and 3, 21.9 days in test group 2).
These findings 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.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

All sperm positive rats got pregnant with one exception in test group 3 (300 mg/kg bw/d).
Female animal no. 135 (test group 3) which was mated with male no. 35 did get sperm in vaginal smear but showed no implants.
The female fertility index was 100% in test groups 0, 1 and 2 and 90% in test group 3.
The gestation index was 100% in all test groups.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see below
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean body weights and body weight change of all male and female pups of all test groups (200, 75 and 300 mg/kg bw/d) were comparable to the concurrent control values throughout the entire lactation period.
Two female runts each were seen in control group and in test group 2 (75 mg/kg bw/d). The amount of runts was well in the range of historical control data (0 to 12 runts) and therefore regarded as incidental and not related to treatment.

Reduction in number of live offspring:

no effects observed

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.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups;
slight differences were regarded to be spontaneous in nature.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.4% / 99.1% / 96.6% and 98.3% in test groups 0, 1, 2, and 3, respectively, showing no association to the treatment.
The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups (0, 1, 2, and 3) respectively without showing any relation to the treatment.

The pup mortality numbers did not show any relation to treatment and were assessed as spontaneous in nature.

Visceral malformations:

no effects observed

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as empty stomach, post mortem autolysis, fluid-filled abdomen, diaphragm hernia and dilated renal pelvis. One pup was partly cannibalized and five pups were not assessed because those were missing (cannibalized).
These findings occurred without any relation to dosing or were observed in a single pup only and considered to be spontaneous in nature.

Other effects:

no effects observed

Description (incidence and severity):

Clinical signs:
There were no test substance-related, adverse clinical signs observed in any of the F1 generation pups of the different test groups.
In control group and test group 1 each on male and one female pup, in test group 2 - 6 male pups and 2 female pups and in test group 3 one male and 3 female pups, were not assessed (died) on one day due to being stillborn, found dead or missing (cannibalized).
This is regarded to be incidental and spontaneous in nature.

- Anogenital distance/anogenital index:
Anogenital distance and anogenital index were significantly decreased in male pups of test group 2 (-5.29% and -4.43%, respectively). Furthermore, anogenital index was significantly reduced in males of test group 3 (-4.71%). All determined anogenital distances and indices were still in the range of historical control data (anogenital distance: 2.91 – 3.36 and anogenital index: 1.52 – 1.75) and therefore regarded as incidental and not related to treatment.
Results on the anogenital index from the study are given in table 6a and 6b.

- Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not significantly influenced by the test substance when examined on PND 13.
The nipple development [%] was increased in test group 1 (20 mg/kg bw/d; 44.9%) and in test group 2 (75 mg/kg bw/d; 14.4%). The nipple number was increased in all test groups (20, 75 and 300 mg/kg bw/d; 53.6%, 42% and 31.9%) to a count of 2.9, 2.6 and 2.5.
These findings showed no statistically significant difference to the current control and no dose-dependency and were assessed as incidental and not related to treatment.

- Thyroid hormones: In male and female pups at PND13 (test groups 11, 12 and 13; 20, 75 and 300 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

no

Table 6a: Summary pup report of anogenital distance 

Phase:Lactation Pup
		Test Group 0/ F	Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d	20 mg/kg bw/d	75 mg/kg bw/d	300 mg/kg bw/d
AG Distance	Mean	3.16 n	3.05	2.99 *	3.01
[mm]	S.d.	0.24	0.06	0.12	0.09
day 1 Males	N	9	10	10	9
	Deviation Vs Control [%]		-3.66	-5.29	-4.78
AG Distance	Mean	1.52 n	1.46	1.44	1.48
[mm]	S.d.	0.14	0.05	0.06	0.02
day 1 Females	N	9	10	10	9
	Deviation Vs Control [%]		-4.08	-5.29	-2.57
AG Distance	Mean	2.38 n	2.25	2.26	2.17
[mm]	S.d.	0.32	0.16	0.16	0.12
day 1 Males + Females	N	9	10	10	9
	Deviation Vs Control [%]		-5.10	-4.85	-8.84

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics n=DUNNETT

 

Table 6b: Summary pup report of anogenital index 

Phase:Lactation Pup
		Test Group 0/ F	Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d	20 mg/kg bw/d	75 mg/kg bw/d	300 mg/kg bw/d
AG Index Cubic Root	Mean	1.67 n	1.63	1.60 *	1.59 *
[---]	S.d.	0.11	0.04	0.05	0.04
day 1 Males	N	9	10	10	9
	Deviation Vs Control [%]		-2.84	-4.43	-4.71
AG Index Cubic Root	Mean	0.82 n	0.79	0.78	0.80
[---]	S.d.	0.06	0.02	0.02	0.02
day 1 Females	N	9	10	10	9
	Deviation Vs Control [%]		-3.54	-4.37	-2.39
AG Index Cubic Root	Mean	1.26 n	1.21	1.21	1.16
[---]	S.d.	0.16	0.10	0.10	0.09
day 1 Males + Females	N	9	10	10	9
	Deviation Vs Control [%]		-4.30	-3.89	-8.54

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics n=DUNNETT

The various analyses confirmed

·     no chemical and microbiological contaminatations in the diet in accordance with Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA),,

·     no analytical findings in drinking water in accordance with the German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990),

·     stability of the test substance in deionized water was demonstrated for a period of 7 days at room temperature,

·     the homogeneous distribution of the test substance in the vehicle,

·     the correctness of the prepared concentrations.

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no treatment-related adverse findings in parental animals (F0) and pups (F1).
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity as well as for reproductive performance and fertility was the highest tested dose 300 mg/kg bw/d in both sexes of parental animals.
The NOAEL for developmental toxicity in the F1 progeny was the highest tested dose 300 mg/kg bw/d.

Executive summary:

The test substance was administered daily as liquid preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at nominal doses of 20, 75 and 300 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 (deionized water). The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 1 day post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females up to one day prior to the

day of schedule sacrifice of the animals.

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

Test group 3: 300 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS: Clinical examinations/ gross findings

- No test substance-related adverse findings

Test group 2: 75 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS: Clinical examinations/ gross findings

- No test substance-related adverse findings

Test group 1: 20 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS: Clinical examinations/ gross findings

- No test substance-related adverse findings

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no treatment-related adverse findings in parental animals (F0) and pups (F1).

The NOAEL for developmental toxicity in the F1 progeny was the highest tested dose 300 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, 55116 Mainz, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

EC Number:

308-662-5

EC Name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Cas Number:

98171-53-0

IUPAC Name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Test material form:

solid

Details on test material:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate amount of test substance was weighed out depending on the desired concentration. Ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were prepared in such a manner that the stability was guaranteed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in ultra-pure water

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

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, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 11-12 weeks (male animals); about 10 weeks (female animals)
- Weight at study initiation: males: 368.3 g (mean); females: 215.2 g (mean)
The weight variation of the animals used did not exceed 20 % of the mean weight of each sex.
- Housing:
- during pretreatment: housed together (up to 5 animals per sex and cage)
- druing study period: individually

- Diet: ad libitum; ground Kliba maintenance diet mouse/rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- 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 was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE.

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

IN-LIFE DATES: From: 19 Sep 2017 To: 08 Nov 2017 (males); 05 Dec 2017 (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least.
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with deionized water and mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.20, 0.75, 3.00 g/100 mL
- Amount of vehicle: 10 mL/kg bw/d
- Purity: deionized water

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as gestation day GD0
- Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
- After successful mating each pregnant female was caged individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop- Rauxel, Germany with nesting material (cellulose wadding) towards the end of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above-mentioned time points, additionally, one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample (described by the suffix “R”) was retained.
The samples collected at the beginning and during lactation period were analyzed in the Analytical Laboratory. Additionally, the reserve samples of the lowest concentration collected at the beginning of the study were analyzed. All further samples were not analyzed and stored frozen (at -20 °C) in the Laboratory of the Mechanistic Toxicology until the finalization of the study.

The reserve samples (sample no. 3R to 5R, mean 96%) of the low dose of premating period were measured due to slightly too high concentration in the primary samples (sample no. 3 to 5, mean 111%) considering the specification of the test facility (90-110%). No deviation was observed in the samples of the low dose (sample no. 26 to 28, mean 108%) collected towards the end of the study.

Thereby, all concentration analyses confirmed the nominal concentration in the test substance preparations. All analytical values were within the specification of the test facility: 90-110% of the nominal concentration.
Considering the three samples collected from one test substance preparation (samples no. 3-5, 3R-5R, 7-9, 26-28, or 30-32) only minor differences were observed within the three samples.

Therefore, the test item was considered as homogeneously distributed in the
vehicle (see Part III, Supplement).

Duration of treatment / exposure:

males: 29 days; females: 56 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

Once daily at approximately the same time in the morning
(Females in labor were not treated.)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

low-dose group

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

mid-level group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the corresponding range finding experiment (repeated dose toxicity study, 14 days, 01R0407/16R119) signs of systematic toxicity were observed at 300 mg/kg bw/d (i.a. respiration sounds, plough nose-first into bedding, and piloerection) and at 1000 mg/kg bw/d (i.a. mortality). The substance appeared to be irritating to the stomach of the mid and high dose group in the study.These findings are in accordance with the classification for skin and eye irritation based on the representative studies.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration
- parturition and lactation behavior of the dams: in the mornings in combination with the daily clinical inspection
- on weekday afternoons (except Saturday, Sunday and public holidays): parturition behavior of the dams
- pups: examined daily for clinical symptoms (including gross-morphological findings)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration and at weekly intervals during administration
- Examined parameters: 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 for female parental animals:
• 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
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.
- Pups: weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13
- individual pup weights: determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week for male and female parental animals
- exceptions:
• 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, 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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes, see table 1
- Time schedule for collection of blood: at termination, study day 29 (males); at PND 14, from study day 50 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.
- blood smears prerared and stained according to WRIGHT, not evaluated because of non-ambiguous results of the differential blood cell counts

CLINICAL CHEMISTRY: Yes, see table 2
- Time schedule for collection of blood: at termination, study day 29 (males); at PND 14, from study day 50 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- functional observational battery performed 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 (Male: study day 27; Female: study day 55) starting at about 10.00 h in a randomized sequence
- Parameters: passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests
- Ranking: according to the degree of severity, if applicable
- Examined observations:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• 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 (auditory 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, other findings

Motor activity assessment:
- Time schedule: at the end of the administration period rom 14:00 h onwards on the same day as the FOB was performed
- Animals: the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Method: TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany

Oestrous cyclicity (parental animals):

Estrous cycle:
- Time schedule: daily analysis of vaginal smear for a minimum of 2 weeks prior to mating, continued throughout the pairing period until the female exhibited evidence of copulation, at necropsy
- Animals: all F0 female parental rats

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations:
- testis weight, epididymis weight, stages of spermatogenesis and histopathology of
interstitial testicular cell structure
- male mating index (number of males with confirmed mating* / number of males placed with females), * = 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), ** = a female with implants in utero

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups or 2 preferably pups/litter were killed and discarded.
- If individual litters did not have 4 pups/sex: litters were processed that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- check for dead or moribund pups: 2x/d on workdays (once in the morning and once in the afternoon)
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities, organs assessed macroscopically; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY, see table 3
- on PND 13, blood sampling for determination of thyroid hormone concentration
- thyroid glands/parathyroid glands fixed and pathology examinations

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, F0 on study day 29
- Maternal animals: All surviving animals, after weaning of pups the female animals were sacrificed

GROSS NECROPSY
- Gross necropsy consisted of gross pathology, special attention being given to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at different stages:
- PND4: surplus pups were sacrificed, blood sampled for determination of thyroid hormone concentrations and examined externally and eviscerated, and the organs were assessed macroscopically
- PND13: one selected male and one female pup per litter sacrificed, lood sampled for determination of thyroid hormone concentrations and thyroid glands/parathyroid glands were fixed for pathology examinations
- remaining pups PND13: sacrificed and examined externally, eviscerated and their organs were assessed macroscopically

GROSS NECROPSY
- Gross necropsy consisted of external and internal abnormalities, organs assessed macroscopically

Statistics:

see table 5

Reproductive indices:

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 utero

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

Offspring viability indices:

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During pre-mating slight salivation after treatment was seen in one male and one female animal on day 4 and piloerection was seen in two males and two females of test group 3 (300 mg/kg bw/d) for up to 3 consequent days. During mating slight to moderate salivation after treatment was seen in two males of test group 3 up to 4 consequent days. During postmating moderate salivation after treatment was seen in two males on post-mating day 0. During gestation alopecia was observed in each one female animal of control group, test group 1 and 2 (20 and 75 mg/kg bw/d) and slight salivation after treatment was observed in two females of test group 3 (300 mg/kg bw/d) on up to 3 consequent days. During the lactation period alopecia still was seen in the same three animals of control group, test group 1 and 2. Furthermore, slight salivation after treatment was seen in three females of test group 3 on single days and one female of test group 3 showed respiration sounds on lactation day 0.
The salivation observed only after treatment at any time point the study were assessed as treatment-related but considered to be caused by bad taste or local irritation. Therefore, it was assessed as non-adverse.

A swelling of the mammary line was observed in three females of control group, four females of test group 1 and each in one female of test groups 2 and 3 on lactation day 14. This finding showed no significant difference to control and no dose-dependency. It was considered as incidental and likely to be a side effect of weaning the litter on PND 13.

Due to animals with delivery in the afternoon gaps were existing for gestation day 22 for two animals (animal nos. 120 and 140) and for lactation day 0 for eight animals (animal nos. 103, 108, 120, 121, 128, 132, 137, 140). These gaps are caused by the change of the phase (from gestation to lactation) within one day not by missing clinical observations.

All findings were considered to be treatment-related but not adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- mean body weights of all male and female F0 generation parental animals in all test substance-treated groups: comparable to the concurrent control values
- exception: males of test group 3 (300 mg/kg bw/d) had significantly decreased body weight on pre-mating day 13 (-4.5%), not observed on studies days thereafter > assessed as treatment-related but not adverse
- bw changes significantly decreased: in male animals of test group 3 (-79.1%), test group 2 (75 mg/kg bw/d; -34.3%) during premating, not observed on studies days thereafter > assessed as treatment-related but not adverse
- changes seen in males of test group 1 (20 mg/kg bw/d; -34.2%) during premating, not observed on studies days thereafter > assessed as treatment-related but not adverse

Food efficiency:

no effects observed

Description (incidence and severity):

Food consumption of the F0 animals in all test groups was not influenced by the treatment throughout the entire study period.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Haematological findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All observations were comparable to control animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia seen in one female animal each in test groups 2 and 1 as well as the control group was not considered to be test substance-related.

In males of test groups 1 (20 mg/kg bw/d), 2 (75 mg/kg bw/d) and 3 (300 mg/kg bw/d) number of rearings was reduced (-35%, -55% and -39%) and in females of test groups 1 and 3 number of rearings was increased (43% and 27%).
Since these findings were neither statistically significant nor dose-dependent altered, they were not considered to be treatment-related.

In female animals of test group 3 (300 mg/kg bw/d) a significantly decrease of beam interruptions was recorded for interval 8. However, since this finding was an isolated single event and no further significant changes were observed in the respective sum of all intervals, this finding was regarded incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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.

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance and anogenital index were significantly decreased in male pups of test group 2 (-5.29% and -4.43%, respectively). Furthermore, anogenital index was significantly reduced in males of test group 3 (-4.71%).
All determined anogenital distances and indices were still in the range of historical control data (anogenital distance: 2.91 – 3.36 and anogenital index: 1.52 – 1.75) and therefore regarded as incidental and not related to treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the parental females of all test groups including the control.
The mean estrous cycle duration in test groups 0, 1 and 3 (0, 20 and 300 mg/kg bw/d) was 4 days and in test group 2 (75 mg/kg bw/d) it was 3.9 days.
The mean number of cycles was 2.5 in test groups 1 and 2 and 2.6 in control group and test group 3.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Fertility was proven for nearly all of the F0 parental males within the scheduled mating interval to produce F1 litter with exception of one male of the control group and one male in test group 3 (300 mg/kg bw/d). These males of control group (no. 6 mated with female no. 106) and of test group 3 (no. 35 mated with female no. 135) did not generate implants in utero.
The male fertility index was 100% in test groups 1 and 2 and 90% in control group and test group 3, see table 6.

The male mating index was 90% in control group and 100% in all test groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

The rate live birth indices were 99.1% in test group 0, 99.2% in test group 1 (20 mg/kg bw/d), 96.8% in test group 2 (75 mg/kg bw/d) and 98.1% in test group 3 (300 mg/kg bw/d). There was 1 stillborn pup recorded in control group and test group 1, 4 stillborn pups recorded in test group 2 and 2 stillborn pups recorded in test group 3. The values of test group 1, 2 and 3 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.

The postimplantation loss was 6.3% in test group 0, 3.3% in test group 1 (20 mg/kg bw/d), 2.4% in test group 2 (75 mg/kg bw/d) and 5.3% in test group 3 (300 mg/kg bw/d).
These findings 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 (0.0% - 18.1%) of the historical control data.

Details on results (P0)

Further information are given in table 7 (overall delivery report) and table 8 (summary of pregnancy).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related, adverse clinical signs observed in any of the F1 generation pups of the different test groups.
In control group and test group 1 each on male and one female pup, in test group 2 - 6 male pups and 2 female pups and in test group 3 one male and 3 female pups, were not assessed (died) on one day due to being stillborn, found dead or missing (cannibalized). Pup status is shown in table 9a and 9b.
This is regarded to be incidental and spontaneous in nature.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.4% / 99.1% / 96.6% and 98.3% in test groups 0, 1, 2, and 3, respectively, showing no association to the treatment.
The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups (0, 1, 2, and 3) respectively without showing any relation to the treatment.

The pup mortality numbers did not show any relation to treatment and were assessed as spontaneous in nature.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight change of all male and female pups of all test groups (200, 75 and 300 mg/kg bw/d) were comparable to the concurrent control values throughout the entire lactation period.
Two female runts each were seen in control group and in test group 2 (75 mg/kg bw/d). The amount of runts was well in the range of historical control data (0 to 12 runts) and therefore regarded as incidental and not related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as empty stomach, post mortem autolysis, fluid-filled abdomen, diaphragm hernia and dilated renal pelvis. One pup was partly cannibalized and five pups were not assessed because those were missing (cannibalized).
These findings occurred without any relation to dosing or were observed in a single pup only and considered to be spontaneous in nature.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Anogenital distance/anogenital index:
Anogenital distance and anogenital index were significantly decreased in male pups of test group 2 (-5.29% and -4.43%, respectively). Furthermore, anogenital index was significantly reduced in males of test group 3 (-4.71%). All determined anogenital distances and indices were still in the range of historical control data (anogenital distance: 2.91 – 3.36 and anogenital index: 1.52 – 1.75) and therefore regarded as incidental and not related to treatment.

- Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not significantly influenced by the test substance when examined on PND 13.
The nipple development [%] was increased in test group 1 (20 mg/kg bw/d; 44.9%) and in test group 2 (75 mg/kg bw/d; 14.4%). The nipple number was increased in all test groups (20, 75 and 300 mg/kg bw/d; 53.6%, 42% and 31.9%) to a count of 2.9, 2.6 and 2.5.
These findings showed no statistically significant difference to the current control and no dose-dependency and were assessed as incidental and not related to treatment.

- Thyroid hormones: In male and female pups at PND13 (test groups 11, 12 and 13; 20, 75 and 300 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

Table 6: Male fertility indices for F0 males

	Test group 0 (0 mg/kg bw/d)	Test group 1 (20 mg/kg bw/d)	Test group 2 (75 mg/kg bw/d)	Test group 3 (300 mg/kg bw/d)
Male fertility index [%]	90	100	100	90

* p ≤ 0.05; ** p ≤ 0.01

Table 7: Mean summary delivery report for all animals and test groups.

Summary Delivery Report   Sex:Female
		Test Group 0/ F 0 mg/kg bw/d		Test Group 1/ F 20 mg/kg bw/d	Test Group 2/ F 75 mg/kg bw/d	Test Group 3/F 300 mg/kg bw/d
No. of females at start	N	10		10	10	10
No. of females mated	N	10	f-	10	10	10
	%	100.0		100.0	100.0	100.0
Pregnant	N	9	f-	10	10	9
	%	90.0		100.0	100.0	90.0
Without delivery	N	1		0	0	1
- Pregnant	N	0		0	0	0
- Not pregnant	N	1		0	0	1
-- Delivering	N	9	f-	10	10	9
	%	100.0		100.0	100.0	100.0
-- With liveborn pups	N	9	f-	10	10	9
Gestation Index	%	100.0		100.0	100.0	100.0
Gestation days	Mean	22.2	n	22.0	21.9	22.0
	S.d.	0.7		0.0	0.3	0.5
	N	9		10	10	9
-- With stillborn pups	N	1	f+	1	2	2
	%	11.1		10.0	20.0	22.2
-- With all pups stillborn	N	0	f+	0	0	0
	%	0.0		0.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics f=FISHER-EXACT; n=DUNNETT

Table 8: Summary pregnancy report with mean values of female groups.

Summary Pregnancy Status Report -   Sex:Female	Reproduction
		Test Group 0/ F, G 10 / F 0 mg/kg bw/d	Test Group 1/ F 20 mg/kg bw/d	Test Group 2/ F 75 mg/kg bw/d	Test Group 3/F 300 mg/kgbw/d
No. of females at start	N	10	10	10	10
No. of females mated	N	10	10	10	10
Without evidence of mating	N	1	0	0	0
- Pregnant	N	0	0	0	0
- Not pregnant	N	1	0	0	0
Females with defined Day 0 pc	N	9	10	10	10
Pregnant	N	9	10	10	9
- sacrificed scheduled	N	9	10	10	9
Not pregnant	N	1	0	0	1
- sacrificed scheduled	N	1	0	0	1
Pregnant, not delivering	N	0	0	0	0
Delivering	N	9	10	10	9
-- With liveborn pups	N	9	10	10	9
	%	100.0	100.0	100.0	100.0
-- With all pups stillborn	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

Table 9.a: Part a of table 9 with the summary litter report, providing the pup status. 

Sex:Female
		Test Group 0/ F 0 mg/kg bw/d		Test Group 1/ F 20 mg/kg bw/d	Test Group 2/ F 75 mg/kg bw/d	Test Group 3/ F 300 mg/kgbw/d
Total Number of Pregnant Females	N	9		10	10	9
Total number of litters	N	9		10	10	9
With liveborn pups	N	9	f-	10	10	9
	%	100.0		100.0	100.0	100.0
With stillborn pups	N	1	f+	1	2	2
	%	11.1		10.0	20.0	22.2
With all pups stillborn	N	0	f+	0	0	0
	%	0.0		0.0	0.0	0.0
Implantation Sites	N	116		129	129	111
	Mean	11.6	x-	12.9	12.9	12.3
	S.d.	4.4		2.5	0.9	1.2
	N	10		10	10	9
Pups delivered	N	108		125	126	105
	Mean	12.0	x-	12.5	12.6	11.7
	S.d.	1.6		2.5	1.2	1.2
	N	9		10	10	9
Postimplantation Loss	Mean%	6.3	x+	3.3	2.4	5.3
	S.d.	9.7		4.6	3.9	5.8
	N	9		10	10	9

Table 9.b: Part b of table 9 with the summary litter report, providing the pup status. 

Sex:Female
		Test Group 0/ F 0 mg/kg bw/d		Test Group 1/ F 20 mg/kg bw/d	Test Group 2/ F 75 mg/kg bw/d	Test Group 3/ F 300 mg/kgbw/d
Pups liveborn	N	107		124	122	103
	%	99.1		99.2	96.8	98.1
	Mean	11.9	x-	12.4	12.2	11.4
	S.d.	1.8		2.5	1.1	1.3
	N	9		10	10	9
Pups stillborn	N	1		1	4	2
	%	0.9		0.8	3.2	1.9
	Mean	0.1	x+	0.1	0.4	0.2
	S.d.	0.3		0.3	1.0	0.4
	N	9		10	10	9
Perinatal Loss	Mean%	1.1	x+	0.8	2.9	1.9
	S.d.	3.3		2.6	6.9	3.8
	N	9		10	10	9

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

The various analyses confirmed

·     no chemical and microbiological contaminatations in the diet in accordance with Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA),,

·     no analytical findings in drinking water in accordance with the German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990),

·     stability of the test substance in deionized water was demonstrated for a period of 7 days at room temperature,

·     the homogeneous distribution of the test substance in the vehicle,

·     the correctness of the prepared concentrations.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no treatment-related adverse findings in parental animals (F0) and pups (F1).
Thus, the no observed adverse effect level (NOAEL) for reproductive performance was the highest tested dose 300 mg/kg bw/d in both sexes of parental animals.
The NOAEL for developmental toxicity in the F1 progeny was the highest tested dose 300 mg/kg bw/d.

Executive summary:

The test substance was administered daily as liquid preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at nominal doses of 20, 75 and 300 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 (deionized water). The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 1 day post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females up to one day prior to the

day of schedule sacrifice of the animals.

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

Test group 3: 300 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS: Clinical examinations/ gross findings

- No test substance-related adverse findings

Test group 2: 75 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS: Clinical examinations/ gross findings

- No test substance-related adverse findings

Test group 1: 20 mg/kg bw/d

F0 PARENTAL ANIMALS: Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS: Clinical examinations/ gross findings

- No test substance-related adverse findings

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no treatment-related adverse findings in parental animals (F0) and pups (F1).

Thus, the no observed adverse effect level (NOAEL) for reproductive performance and fertility was the highest tested dose 300 mg/kg bw/d in both sexes of parental animals.