Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
In addition to the standard OECD 421 protocol, functional observational battery (FOB) as well as motor activity measurements (MA) were performed in all animals.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris(2-hydroxyethyl)methylammonium methyl sulphate- Physical state: liquid- Analytical purity: 98.9% by weight- Lot/batch No.: Lot. 29734916K0- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor andthe sponsor holds this responsibility.- Storage condition of test material: room temperature; avoid temperatures <5°C and >30°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories, Research Models andServices, Germany GmbH- Age at study initiation: 10-11 weeks; males/females- Weight at study initiation: animals of comparable size and weight- Housing: During the study period, the rats were housed individually in Polycarbonate type M IIIcages supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co.,Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions• During overnight matings, male and female mating partners were housed togetherin Polycarbonate type M III cages.• Pregnant animals and their litters were housed together until PND 4 (end oflactation).• For motor activity (MA) measurements the animals were housed individually inpolycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany, withwire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2), andsmall amounts of bedding materialPregnant females were provided with nesting material (cellulose wadding) toward the endof gestation.Dust-free wooden bedding was used in this study (the present supplier is documented inthe raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. andVet. Service GmbH, Vienna, Austria, was added for environmental enrichment.The cages with the test animals were arranged on the racks in such a way that uniformexperimental conditions (ventilation and light) were ensured.- Diet: ad libitum- Water: ad libitum- Acclimation period: at least 5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24- Humidity (%): 30-70- Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Tris(2-hydroxyethyl)methylammonium methyl sulphate was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least twice a week.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06:30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data.A vaginal smear was prepared after each mating and examined for the presence of sperm.If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany under the responsibility of the Study Director of this test facility.The study was carried out in compliance with the Principles of Good Laboratory Practice.The stability of the test substance in drinking water at room temperature over a period of 4 days was demonstrated before the start of the study (study No. 14L00227; see PART III, Supplement).Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and at the end of the administration period. Given that the test substance is completely miscible with drinking water, solutions were considered to be homogenous and no homogeneity analyses were carried out.
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.
Frequency of treatment:
Continuous via drinking water
Details on study schedule:
N/A
Doses / concentrations
Remarks:
Doses / Concentrations:0 ppm, 1200 ppm, 4000 ppm and 12000 ppmBasis:nominal in water
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
The dose levels were selected based on the results of a range-finding study.
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
MortalityA check for moribund and dead animals was made twice daily on working days and oncedaily on Saturdays, Sundays and public holidays. If animals were in a moribund state, theywere sacrificed and necropsied.Clinical observationsA cageside examination was conducted at least once daily for any signs of morbidity,pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes weredocumented daily for each affected animal.The littering and lactation behavior of the dams was generally evaluated in the mornings incombination with the daily clinical inspection of the dams. Only particular findings (e.g.inability to deliver) were documented on an individual dam basis.On weekdays (except public holidays) the parturition behavior of the dams was inspectedin the afternoons in addition to the evaluations in the mornings.The day of littering was considered the 24-hour period from about 15.00 h of one day untilabout 15.00 h of the following day.Detailed clinical observationsDetailed clinical observations (DCO) were performed in all animals prior to theadministration period and thereafter at weekly intervals. The findings were rankedaccording to the degree of severity, if applicable. The animals were transferred to astandard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters wereexamined:1. abnormal behavior in handling2. fur3. skin4. posture5. salivation6. respiration7. activity/arousal level8. tremors9. convulsions10. abnormal movements11. gait abnormalities12. lacrimation13. palpebral closure14. exophthalmos15. assessment of the feces discharged during the examination (appearance/consistency)16. assessment of the urine discharged during the examination17. pupil sizeWater consumptionGenerally, water consumption was determined once a week for male and female parentalanimals, with the following exceptions:• Water consumption was not determined during the mating period (male and female F0animals).• Water consumption of the F0 females with evidence of sperm was determined on GD6-7, 13-14 and 19-20.• Water consumption of F0 females, which gave birth to a litter was determined forPND 3-4.Water consumption was not determined in females without positive evidence of spermduring mating and gestation periods and in females without litter during the lactation periodand in males after the premating period.Food consumptionGenerally, food consumption was determined once a week for male and female parentalanimals, with the following exceptions:• Food consumption was not determined during the mating period (male and female F0animals).• Food consumption of the F0 females with evidence of sperm was determined on GD0-7, 7-14 and 14-20.• Food consumption of F0 females, which gave birth to a litter was determined forPND 1-4.Food consumption was not determined in females without positive evidence of sperm(during the mating period of dams used in parallel) and females without litter (during thelactation period of dams used in parallel) and in males after the premating period.Body weight dataBody weight was determined before the start of the administration period in order torandomize the animals. During the administration period body weight was determined onstudy day 0 (start of the administration period) and thereafter once a week at the sametime of the day (in the morning).The body weight change of the animals was calculated from these results.The following exceptions are notable for the female animals:• During the mating period the parental females were weighed on the day of positiveevidence 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.• Females without a litter and without positive evidence of sperm in the vaginal smear orwaiting for necropsy were weighed weekly and these values are only documented inthe Individual Tables (PART II).Intake of test substanceThe mean daily intake of test substance (group means) was calculated based uponindividual values for body weight and mean water consumption per cage.To calculate the mean values of entire administration period for each test group, thevalues of each study period, i.e. premating (14 days), gestation (20 days) and lactation (4days), were assessed according to days of treatment.Functional observational batteryA functional observational battery was performed in the first five surviving male animalsper test group and the first five surviving females with litter (in order of delivery) of all testgroups at the end of the administration period starting at about 10.00 h. The FOB startedwith passive observations without disturbing the animals, followed by removal from thehome cage, open field observations in a standard arena and sensorimotor tests as well asreflex tests. The findings were ranked according to the degree of severity, if applicable.The observations were performed at random. A detailed description of the methods, theranking and documentation system can be found in PART III (Supplement).Home cage observations:The animals were observed in their closed home cages; during this period any disturbingactivities (touching the cage or rack, noise) were avoided during these examinations inorder not to influence the behavior of the animals. Attention was paid to:1. Posture2. Tremors3. Convulsions4. Abnormal movements5. Impairment of gait6. Other findingsOpen field observations:The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height)and observed for at least 2 minutes. The following parameters were examined:1. behavior on removal from the cage2. fur3. skin4. salivation5. nose discharge6. lacrimation7. eyes/pupilsize8. posture9. palpebral closure10. respiration11. tremors12. convulsions13. abnormal movements/stereotypes14. gait abnormalities15. activity/arousal level16. feces excreted within 2 minutes (appearance/consistency)17. urine excreted within 2 minutes (amount/color)18. rearing within 2 minutes19. other findingsSensory motor tests/ reflexes:The animals were then removed from the open field and subjected to following sensorymotor or reflex tests:1. Reaction to an object being moved towards the face (approach response)2. touch sensitivity (touch response)3. vision (visual placing response)4. pupillary reflex5. pinna reflex6. audition (auditory startle response)7. coordination of movements (righting response)8. behavior during handling9. vocalization10. pain perception (tail pinch)11. other findings12. grip strength of forelimbs13. grip strength of hindlimbs14. landing foot-splay testMotor activity assessmentMotor activity (MA) was also measured from 14:00 h onwards on the same day as theFOB was performed in the first five parental males and the first five surviving females withlitter (in order of delivery) per group. The examinations were performed using the TSELabmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For thispurpose, the animals were placed in new clean polycarbonate cages with a small amountof bedding for the duration of the measurement. Eighteen beams were allocated per cage.The numbers of beam interrupts was counted over 12 intervals of 5 minutes per interval.The sequence in which the animals were placed in the cages was selected at random. Onaccount of the time needed to place the rats in the cages, the starting time was"staggered" for each animal. The measurement period began when the 1st beam wasinterrupted and finished exactly 1 hour later. No food or water was offered to the animalsduring these measurements and the measurement room was darkened after the transferof the last animal. The program required a file name for the measured data to be stored.This name consisted of the reference number and a serial number.Male reproduction dataThe pairing partners, the number of mating days until vaginal sperm was detected in thefemale animals, and the gestational status of the females were recorded for F0 breedingpairs.For the males, mating and fertility indices were calculated for F1 litters.Female reproduction and delivery dataThe pairing partners, the number of mating days until vaginal sperm were detected andgestational status were recorded for F0 females.For the females, mating, fertility and gestation indices were calculated for F1 litters.The total number of pups delivered and the number of liveborn and stillborn pups werenoted, and the live birth index was calculated for F1 litters.The implantations were counted and the postimplantation loss (in %) was calculated.To determine the number of implantation sites, the apparently non-pregnant uteri werestained for about 5 minutes in 1% ammonium sulfide solution according to the method ofSALEWSKI (Salewski, E.; 1964). Then the uteri were rinsed carefully in physiologic saltsolution (0.9% NaCl). Thereafter the implantation sites were recorded for calculation of thepostimplantation loss.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
N/A
Litter observations:
Litter/PupsLitter dataPup number and status at deliveryAll pups delivered from the F0 parents (F1 litter) were examined as soon as possible onthe day of birth to determine the total number of pups, the sex and the number of livebornand stillborn pups in each litter. At the same time, the pups were also being examined formacroscopically evident changes. Pups, which died before this initial examination, weredefined as stillborn pups.Pup viability/mortalityIn general, a check was made for any dead or moribund pups twice daily on workdays(once in the morning and once in the afternoon) or as a rule, only in the morning onSaturdays, Sundays or public holidays.The number and percentage of dead pups on the day of birth (PND 0) and of pups dyingbetween PND 1-4 (lactation period) were determined. Pups which died accidentally orwere sacrificed due to maternal death were not included in these calculations. The numberof live pups/litter was calculated on the day after birth, and on lactation day 4. The viabilityindex was calculated.Sex ratioOn the day of birth (PND 0) the sex of the pups was determined by observing the distancebetween the anus and the base of the genital tubercle; normally, the anogenital distance isconsiderably greater in male than in female pups. The sex of the pups was finallyconfirmed at necropsy.The sex ratio was calculated at day 0 and day 4 after birth.Pup clinical observationsThe live pups were examined daily for clinical symptoms (including gross-morphologicalfindings) during the clinical inspection of the dams.Pup body weight dataThe pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weightchange was calculated from these results. The individual weights were always determinedat about the same time of the day (in the morning). In the summary tables pup bodyweights and pup body weight change are listed for males, females and males + females.“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups thatweigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
PATHOLOGYNecropsyAll parental animals were sacrificed by decapitation under isoflurane anesthesia. Theexsanguinated animals were necropsied and assessed by gross pathology, specialattention being given to the reproductive organs.Organ weightsThe following weights were determined in all animals sacrificed on schedule:1. Anesthetized animals2. Epididymides3. Testes4. KidneysOrgan/tissue fixationThe following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution orin modified Davidson’s solution:1. All gross lesions2. Cervix3. Coagulating glands4. Epididymides (modified Davidson’s solution)5. Ovaries (modified Davidson’s solution)6. Oviducts7. Prostate gland8. Seminal vesicles9. Testes (modified Davidson’s solution)10.Vagina11.Uterus (uteri of all cohabited female F0 parental animals were stained according toSalewski’s method: see chapter 3.8.1.1112.KidneysHistopathologyFixation was followed by histotechnical processing, examination by light microscopy andassessment of findings in the following organs in all the control and high dose group animals:TestesEpididymidesOvariesThe organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).A correlation between gross lesions and histopathological findings was attempted.
Postmortem examinations (offspring):
Pup necropsy observationsAll pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesiawith CO2. All pups were examined externally and eviscerated; their organs were assessedmacroscopically.All stillborn pups and all pups that died before PND 4 were examined externally,eviscerated and their organs were assessed macroscopically.All pups without notable findings or abnormalities were discarded after their macroscopicevaluation. Animals with notable findings or abnormalities were evaluated on a case-bycasebasis, depending on the type of finding noted.
Statistics:
Detailed statisitical analyses were conducted as indicated in the study report.
Reproductive indices:
see parental animals: Observations and examinations
Offspring viability indices:
see litter observations

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

MortalityNo animal died prematurely in the present study.Summary clinical observations for males and femalesNo clinical findings were observed in animals during premating, mating and post mating periods.Summary clinical observations for females during gestationNo clinical findings were observed for female animals of test groups 0-3 (0, 1200, 4000 and 12000 ppm).Clinical observations for females during lactationFemale animal No. 112 of test group 1 (1200 ppm) showed a skin lesion at both flanksfrom lactation days 11 to 18. On lactation day 18 another skin lesion in the neck regionwas also observed.Female animal No. 123 of test group 2 (4000 ppm) showed also a skin lesion in the neckregion from lactation days 13 to 19.The findings in both animals were assessed to be incidental and not related to treatment.No other clinical findings were observed for female animals of test groups 0-3 (0, 1200,4000 and 12000 ppm).Water consumptionWater consumption was significantly increased in males of test group 3 (12000 ppm) onpremating days 7 (+21%) and 13 (+43%). In females of test group 3 water consumptionwas significantly increased on premating day 13 (+35%). The reason for this effect couldnot be elucidated.Food consumptionFood consumption in females of test group 2 (4000 ppm) was significantly increased onpremating day 7 (+8.8%) and on gestation day 7 (+8.1%). A relation to dosing was notassumed.No comparable observation were made in any other test group of the study.Body weight dataNo significant deviation in mean body weights and body weight changes were observed forall animals in all test groups.Intake of test substanceThe intake of Tris(2-hydroxyethyl)methylammonium methyl sulphate was calculated on thebasis of most recent individual body weights in each test group.Males: 0, 75, 253 and 774 mg/kg bw/day.Females: 0, 118, 439 and 1564 mg/kg bw/day.Detailed clinical observationsFemale animal Nos. 112 and 123 of test groups 1 (1200 ppm) and 2 (4000 ppm),respectively, showed skin lesions during DCO on study day 49. The findings in bothanimals were assessed to be incidental and not related to treatment.No other clinical findings were observed for males and female animals of test groups 0-3(0, 1200, 4000 and 12000 ppm).Functional observational batteryDeviations from "zero values" were obtained in several rats. However, as most findingswere equally distributed between test-substance treated groups and controls, without adose-response relationship or occurred in single rats only, these observations wereconsidered as incidental.The following examinations were performed during FOB and are assessed individually:Home cage observationsNo test substance-related effects were observed.Open field observationsFemale animal Nos. 112 and 123 showed skin lesions during open field observations. Notest substance-related effects were observed.Sensorimotor tests/reflexesNo test substance-related effects were observed.Quantitative ParametersNo test substance-related effects were observed.Motor activity measurementThere were no significant deviations concerning the overall motor activity (summation of allintervals) in male and female animals of all test groups in comparison to the concurrentcontrol group.Regarding single intervals in female animals of test group 1 (1200 ppm) a significantlyreduced activity was observed in interval 10. As no other single intervals as well as theoverall motor activity showed significant deviations to the control values, the finding wasassessed to be incidental and not related to treatment.Male reproduction data Male mating indexThe male mating index was 100% in test group 0 and 1 and 90% in test groups 2 and 3 asfemale animal Nos. 125 of test group 2 and 134 of test group 3 were not inseminated (nosperm was detected in the vaginal smear).Male fertility indexFertility was proven for most of the F0 parental males within the scheduled mating intervalto produce F1 litter.Male animal Nos. 13 and 15 of test group 1 (1200 ppm), which were mated with femaleanimal Nos. 113 and 115, and male No. 25 of test group 2 (4000 ppm), which was matedwith female No. 125, as well as male animal No. 34 of test group 3 (12000 ppm), whichwas mated with female No. 134, did not generate F1 pups.Thus, the male fertility index was 80% in test group 1 and 90% in test groups 2 and 3. These values reflected the normal range of biological variationinherent in the strain of rats used for this study as all respective values were within therange of the historical control data.Female reproduction and delivery dataFemale mating indexThe female mating index calculated after the mating period for F1 litter was 100% in testgroups 0 and 1 and 90% in test groups 2 and 3.The mean duration until sperm was detected (GD 0) was 2.0 days for test group 0, 3.6days for test group 1, 2.4 days for test group 2 and 1.8 days for test group 3. These valuesreflected the normal range of biological variation inherent in the strain of rats used for thisstudy as all respective values were within the range of the historical control data.Female fertility indexAll sperm positive rats delivered pups with two exceptions in test group 1 (1200 ppm).Female animal Nos. 113, which was mated with male No. 13, and 115, which was matedwith male No. 15, had sperm in vaginal smear but delivered no pups and showed noimplants.Thus, the female fertility index was 100% in test groups 0, 2 and 3 and 80% in test group 1.These values reflected the normal range of biological variation inherent in the strain of rats.The mean duration of gestation was similar in all test groups, i.e. 22..2 days (test group 0,1 and 2) and 22.1 days in test group 3 These findings reflected the normal range ofbiological variation inherent in the strain of rats used for this study as all respective valueswere within the range of the historical control data.Gestation indexThe gestation index was 100% in all test groups.Live birth indicesThe rate live birth indices were 100% in all test groups.Postimplantation lossThe postimplantation loss was 7.4% in test group 0 (control), 12.8% in test group 1 (1200ppm), 1.0% in test group 2 (4000 ppm) and 3.1% in test group 3 (12000 ppm). Thesevalues reflected the normal range of biological variation inherent in the strain of rats usedfor this study as all respective values were within the range of the historical control data.PATHOLOGYAbsolute and relative weightsAbsolute organ weightsAll mean absolute weight parameters did not show significant differences when comparedto the control group 0.Relative organ weightsAll mean relative weight parameters did not show significant differences when comparedto the control group 0.Gross lesionsAll findings occurred either individually or were biologically equally distributed over controland treatment groups. They were considered to be incidental or spontaneous in origin andwithout any relation to treatment.Fertility:The female animals (Nos. 113, 115, 125, and 134), which were not pregnant, as well asthe male mating partners (Nos. 13, 15, 25, and 34) did not show relevant gross lesions.HistopathologyAll findings occurred either individually or were biologically equally distributed over controland treatment groups. They were considered to be incidental or spontaneous in origin andwithout any relation to treatment.FertilityThe female animal No. 134, which was not pregnant, as well as the male mating partnerNo. 34 did not show relevant histopathological findings. Animal Nos. 113, 115, 125 and 13,15, 25 were not investigated histopathologically.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
general systemic toxicity, neurotoxicity and reproductive performance and fertility
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
reproductive performance

Results: F1 generation

Details on results (F1)

F1 generation litter/pupsLitter dataPup number and status at deliveryThe mean number of delivered F1 pups per dam was equally distributed among groups 0,1, 2 and 3. No significant deviations occurred.Pup viability/mortalityThe viability index indicating pup mortality during lactation (PND 0-4) was 100% in testgroups 1, 2 and 3 and 99.3% in test group 0. These findings reflected the normal range ofbiological variation inherent in the strain of rats used for this study as all respective valueswere within the range of the historical control data (PART III, Supplement).One pup of test group 0 (control) was cannibalized. Each one pup in test groups 1 and 2were found stillborn. A relation to treatment was excluded.Sex ratioThe sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did notshow substantial differences between the control and the test substance-treated groups;slight differences were regarded to be spontaneous in nature.Pup clinical observationsOne pup of test group 1 (No. 116-15) was sacrificed moribund because of anal atresia andan absent tail.One female pup of test group 2 (No. 129-12) and one male pup of test group 1 (No.119-07) were found stillborn. One male pup of test group 0 (No. 103-07) was cannibalized onPND 1.All other surviving F1 pups of any test group did not show adverse clinical signs up toscheduled sacrifice on PND 4.Pup body weight dataMean pup body weights/pup body weight changes of all pups in all test groups werecomparable to the control group.One male runt was found in test group 0 and one female runt was found in test group 2.Pup necropsy observationsOne female pup of test group 1 (No. 116-15) showed an absent tail and one male pup oftest group 1 (No. 119-07) showed post mortem autolysis. These findings were assessedas being spontaneous in nature and without toxicological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

ANALYSES

Stability analysis

The stability of the test substance in drinking water was demonstrated over a period of 4

days at room temperature. As the mixtures were stored no longer than this time period, the

stability was guaranteed (project No. 14L00227).

Homogeneity control analyses

Given that the test substance was completely miscible with drinking water, solutions were

considered to be homogenous without further analysis.

Concentration control analysis of the test substance preparations

At the beginning of the administration period (study day 0: 23 Feb 2015) samples 3 - 5

were taken and send to the analytical laboratory (project No. 15L00112). The values of

sample numbers 3 and 4 were found to be in the range of 90-110% of the nominal

concentrations (see Part III, Supplement). The concentrations of sample No. 5 (64%) and

5R (retain sample, 56%) were not in good accordance with the expected concentration.

Towards the end of the administration period (study day 52: 16 Apr 2015) another

analytical examination was carried out. Samples 8 - 10 were taken and sent to the

analytical laboratory (project No. 15L00204). The values for each test group were found to

be in the range of 90-110% of the nominal concentrations.

Therefore, the deviations found in samples 5 and 5R were assessed to be incidental.

Food analyses

On the basis of duration of use and the analytical findings with respect to chemical and

microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91

of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical

contaminants. The number of microorganisms did not exceed 1*105/g food. Individual

results can be found in the archives of the Experimental Toxicology and Ecology of

BASF SE.

Drinking water analyses

On the basis of the analytical findings the drinking water was found to be suitable. German

“Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum

tolerable contaminants. Individual results can be found in the archives of the Experimental

Toxicology and Ecology of BASF SE.

Bedding and enrichment analyses

On the basis of the analytical findings the bedding and the enrichment are found to be

suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for

maximum tolerable contaminants. Individual results are to be found in the archives of the

Experimental Toxicology and Ecology of BASF SE.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present Reproduction/Developmental Toxicity Screening Testthe oral administration of Tris(2-hydroxyethyl)methylammonium methyl sulphate to maleand female Wistar rats did not result in signs of systemic toxicity up to a concentration of12000 ppm. Thus, the no observed adverse effect level (NOAEL) for general systemictoxicity was 12000 ppm for male (774 mg/kg bw/d) and female Wistar rats (1564 mg/kgbw/d).In addition, signs of neurotoxicity were not observed. The NOAEL for neurotoxicity was setto 12000 ppm for male and female animals.The NOAEL for reproductive performance and fertility was also set to 12000 ppm for maleand female Wistar rats.The NOAEL for developmental toxicity was 12000 ppm.
Executive summary:

Tris(2-hydroxyethyl)methylammonium methyl sulphate was administered orally via drinking

water to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 ppm

(test group 0), 1200 ppm (test group 1), 4000 ppm (test group 2) and 12000 ppm (test

group 3).

The duration of treatment covered a 2-week premating and a mating period in both sexes,

approximately 1 week post-mating in males, and the entire gestation period as well as 4

days of lactation and 2 weeks thereafter in females.

Regarding clinical examinations, signs of general systemic toxicity were not observed in

male or female parental animals of test groups 1-3 (1200, 4000 and 12000 ppm) during

the entire study period. In addition, functional observational battery (FOB) as well as motor

activity measurements (MA) did not reveal any signs of a neurotoxic potential.

Fertility indices for male and female animals were not impaired by test-substance

administration even at a concentration of 12000 ppm. In addition, live birth indices of pups

in all test groups were not influenced.

The viability index as indicator for pup mortality was not altered.

Regarding pathology, no treatment-related findings were observed with regard to organ

weights, macroscopic and histologic findings. 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.