Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 2019 - 03 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 2019 - 03 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the design of this study is essentially conform to the following guidelines.
• OECD Guideline 421. Reproduction/Developmental Toxicity Screening Test, July 2016.
• EPA Health Effects Test Guideline OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, July 2000.
• Council Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
• OECD Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
• EPA Health Effects Test Guideline OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
pH: 13 at concentration of 20%
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 10-11 weeks; females: 13-14 weeks
- Weight at study initiation: males: 245 - 299 g; females: 205 - 238 g
- Fasting period before study: F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before the day of scheduled necropsy, but water was available. F0- females were not fasted overnight before necropsy
- Housing: On arrival and following pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. For psychological/environmental enrichment and nesting material, animals were provided with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water ad libitum. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: at least 5 days

It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 32 - 54
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 March 2019 To: 21 May 2019
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For dosing of Group 4 animals: The test item was administered as a solution as received. An adequate amount of the test item was dispensed into daily aliquots, which was stored in a controlled temperature area set to maintain 21°C until use.
For dosing of Group 2 and 3 animals: Test item dosing formulations (w/w) was homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. A factor of 4.92 was used to correct for the purity/composition of the test item.

Dose volume: 0.0717 mL/g
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the high dose group (Group 4), the test item was used as received from the Sponsor; therefore, samples for dose formulation analysis for Group 4 were not collected by the Test Facility.
Samples for Analysis: Duplicate middle samples for Groups 1 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 3 (concentration and homogeneity analysis).
Dose formulation samples were collected during week 1 and week 5 of treatment. Sample Volume: Approximately 500 mg accurately weighed into volumetric flasks of 100 mL which were filled up to mark with water.

Details on analytical veryfication: Analysis was based on the analytical method validated for the test item in vehicle in Charles River project 20165222.

Acceptance Criteria:
For concentration: mean sample concentration results within or equal to ±10% for solutions of target concentration.
For homogeneity: coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Duration of treatment / exposure:
Males were treated for 29 days, females that delivered were treated for 51-55 days, i.e.14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13, 14 or 15 days after delivery. Females who failed to deliver were treated for 42-43 days.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-Day dose range finding study with oral gavage of the test item.
- Rationale for animal assignment: A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
- Fasting period before blood sampling for clinical biochemistry: Males were fasted overnight, females were not fasted.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: F0-animals: Samples were collected, between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only
- How many animals: F0-animals 5/sex/group
- Parameters according to OECD 422 guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: F0-animals: Samples were collected, between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only
- How many animals: F0-animals 5/sex/group
- Parameters according to OECD 422 guideline

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood:
F0-animals: Samples were collected, between 7.00 and 10.30 a.m. on the day of scheduled necropsy
F1-animals: Samples were collected, between 7.00 and 10.30 a.m. on PND 4 at culling and PD14-16 on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only
- How many animals: F0-animals all animals, F1-animals 2 pups/litter (one male and one female, if possible)
- Parameters examined: total T4 in all F0-males and PND 14-16 pups
For the F0-generation, assessment of T4 (females) and TSH (both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 7, 8, 9, 10 and 11). These tests were performed after dosing, after completion of clinical observations.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No

ESTROUS CYCLE DETERMINATION:
- Time schedule for examinations: Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
- Dose groups that were examined: all
- Parameters examined: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ were weighed and tissues collected according to OECD 422 guideline.

HISTOPATHOLOGY: Yes
Tissues were processed and examined according to OECD 422 guideline.

Selected tissues were applicable for males that failed to sire and females that failed to deliver pups (i.e. nonmated females, non-pregnant females, no offspring): Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.

Thymus of males of Groups 2 and 3 were identified by the study pathologist during microscopic evaluation as target tissues and were evaluated and reported.

For the testes of all selected males of Groups 1 and 4, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Parametric (ANOVA) tests on group means with Bonferroni correction for multiple testing) was used for the motor activity data set.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Relevant clinical signs noted for the animals found dead or sacrificed in extremis are specified under Mortality.

In surviving animals, rales and/or gasping were seen incidentally in one female animal treated at 5 mg/kg and two males and one female animal treated at 15 mg/kg. This was considered not toxicologically relevant based on the incidence observed.
Other clinical signs noted during the treatment period included alopecia, wounds and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
No findings were noted during the weekly arena observations in this study.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were four premature decedents in the study, all treated at 15 mg/kg/day.
One male was sacrificed after 8 days of treatment. This animal presented itself with lethargic behaviour, ventro-lateral recumbency, quick breathing and salivation. Furthermore, a bodyweight loss of 6% compared to the day before. At necropsy, this animal showed macroscopically enlarged adrenal glands with red foci, enlarged urinary bladder with red watery-clouded content, enlarged kidneys with red foci and pelvic dilation, dilated ureters, stomach with red foci and gelatinous content, gelatinous seminal vesicles and watery-clear fluid in the abdominal cavity. Microscopically, all findings were related to the presence of a large abscess in the prostate and severe inflammation and necrosis of the urinary bladder and seminal vesicles with consequently retrograde nephritis in the kidney. Most other microscopic findings were likely secondary to this inflammation (i.e. hemorrhage in adrenal glands, histiocytosis and lymphoid depletion in the marginal zone of the spleen, inflammatory cell infiltrate in the seminal vesicles and ureters, increased myeloid cells in the bone marrow). It is unclear whether these uncommon findings are (indirectly) related to the test item.

One female was found dead after 9 days of treatment. The animal was partly cannibalized and organs were severely autolytic. Therefore it was not possible to establish the cause of death.

One female was found dead after 23 days of treatment (Day 8 post-coitum). This animal presented itself with lethargic behaviour, flat and hunched posture and piloerection. Macroscopic findings of note were red-brown gelatinous contents in the jejunum and fluid in the uterus (red-brown), vagina (red-brown) and abdominal cavity (watery clear). Microscopic findings consisted of granulocytic inflammatory cell infiltrates in and around the thymic lymph node (together with aggregates of bacteria), the lung pleura and the ovarian serosa. Luminal blood was present in the uterus (corresponds to red-brown fluid in uterus/vagina). In lung and liver increased numbers of intravascular granulocytes were observed. These acute inflammatory cell infiltration at several sites together with presence of bacteria in/around the thymic lymph node, intravascular increased granulocytes and the macroscopically changes in content or fluid at several locations may indicate sepsis. This is possibly related to the gavage procedure but a relationship to the test item cannot be excluded.

One female was sacrificed after 2 days of treatment. This animal presented itself with laboured respiration, gasping and squeaking. Macroscopic findings of note were red foci in jejunum and ileum (ileum also distended with gas) and irregular surface of the glandular mucosa of the stomach. There was no clear microscopic correlate and there were no microscopic findings that could explain the moribundity of the animal.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.
During post coitum, relative food consumption was slightly lower between Day 14-20 for females treated at 15 mg/kg, reaching statistical significance between Day 14-17 post coitum. Based on the low magnitude of the change and since relative food consumption returned to control levels during the lactation phase, this was considered not toxicologically relevant.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological and coagulation parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in haematology and coagulation parameters were considered to be unrelated to treatment given the minimal magnitude of the change and as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in functional observation parameters were noted.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and treated animals.
For males treated at 15 mg/kg bw/day, the number of total movements and ambulations were 18% and 22% lower, respectively. No statistical significance was achieved and as all values remained within the range of the historical control data1, these changes were considered not to be toxicologically relevant. For females, motor activity was similar between treated and control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one mid dose female, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
There were 3/6 surviving males treated at 15 mg/kg with apoptosis in the thymus up to slight degree compared to 0/5 males in the controls. Intermediate dose groups showed incidences of 2/5 males at 1.5 mg/kg and 0/5 in males treated at 5 mg/kg. Apoptosis in the thymus can be observed as a background finding and based on the low severity and the absence of a dose relationship, the finding is considered not test item-related.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration
in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analyses:
Serum levels of T4 in F0 males were considered not to be affected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
other: BTMAOH
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (actual dose received)
System:
other: Mortality
Organ:
other: based on four early deaths
Treatment related:
yes

Results dose range finder:

A dose range finder was conducted previously to select dose levels for the main study and to determine the peak effect of occurrence of clinical signs after dosing. No testing guidelines were applicable as this dose range finder was intended for dose level selection purposes only.

A dose of 100 mg/kg bw/day was not tolerated by the animals as severe clinical signs, reduced food consumption and mild to severe body weight loss were observed. All animals treated with 100 mg/kg bw/day died before scheduled necropsy on Day 2, 5 or 6 of treatment. At dose level 25 mg/kg bw/day, some clinical symptoms, reduced food consumption and slight body weight loss were observed during the 14 Days of treatment. One animal at 25 mg/kg bw/day died at Day 8 of treatment. A dose of 15 mg/kg bw/day was well tolerated for 28 days and therefore a maximum dose level of 15 mg/kg bw/day was selected for the main study. Because of the high pH of the test item and its corrosive properties, the dose volume for the main study was set at 0.072 mL/kg, based on the dose volume used for the 15 mg/kg bw/day Group of the dose range finder. In the dose range finder, clinical signs were generally observed directly after dosing.

Dose formulation Analyses

Week 1

Accuracy

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

The concentrations analyzed in the formulations of Group 2 were in agreement with target concentrations (98% of target). The mean accuracy of the Group 3 formulation was below the target concentration (i.e. 86% of target), which was probably due to analytical issues. A new formulation analysis was scheduled in Week 5 of the toxicology study.

Week 5

Accuracy

A small response at the retention time of the test item was integrated in one of the chromatograms of the Group 1 formulation, which was assigned to noise since the peak height was within the fluctuation of the baseline. It was considered not to derive from the formulation since the response was not observed in the duplicate sample.

The concentrations analyzed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (mean accuracies 109% and 99%, respectively).

Homogeneity

The formulations of Group 2 and Group 3 were homogeneous (coefficient of variation of 2.8% and 6.1%, respectively).

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20165222) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Conclusions:
In a combined 28-day repeated dose toxicity study with reproduction/developmental toxicity test was performed according to OECD TG 422. The systemic NOAEL was 5 mg/kg bw/day as mortality was observed at 15 mg/kg bw/day. No reproductive or developmental toxicity was observed up to and including the highest dose levels and the NOAEL for reproduction and developmental was at least 15 mg/kg bw/day.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental screening toxicity test was performed according to OECD TG 422 and in accordance with GLP. Wistar rats, 10 per sex/dose were exposed to 1.5, 5 and 15 mg/kg bw/day BTMAOH. The dose levels were selected based on the dose range finder in which mortality was observed at 100 mg/kg bw/day (3/3 animals) and 25 mg/kg bw/day (1/3 animals). Because of the high pH of the test item and its corrosive properties, the dose volume for the main study was set at 0.072 mL/kg. Formulation analyses conducted during the study confirmed that formulations of test item in water were prepared accurately and homogeneously.

Parameters and endpoints evaluated in this study were according to the guideline and included: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0 -males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Four early deaths were observed at 15 mg/kg bw/day: one male and one female animal were sacrificed in extremis and two female animals were found dead. The moribundity of the male animal sacrificed in extremis was most likely caused by a large abscess in the prostate and severe inflammation and necrosis of the urinary bladder and seminal vesicles with consequently retrograde nephritis in the kidney. The moribundity of the female animal sacrificed in extremis could not be explained by the macroscopic and microscopic findings. For the third animal, a female found dead, evaluation showed signs indicating sepsis. The cause of death of the other female found dead could not be established due to cannibalism and autolysis. Although there is no consistency in the morphological findings or the cause of death/moribundity of the four premature deaths and there were no similar morphological findings in the scheduled sacrificed rats, these premature death were considered test-item related given the high incidence in the highest dose group only.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical observations, body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), functional observations, macroscopic examination, organ weights, and microscopic examination).

No reproductive toxicity was observed up to the highest dose level tested (15 mg/kg bw/day).

Due to the three early deaths and two cases of non-pregnancy at 15 mg/kg, there were only five viable litters available for evaluation. No developmental toxicity was observed up to the highest dose level tested (15 mg/kg bw /day).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following NOAELs of Benzyltrimethylammonium hydroxide were established:

Parental NOAEL: 5 mg/kg bw/day (Based on four early deaths at 15 mg/kg bw/day)

Reproduction NOAEL: at least 15 mg/kg bw/day

Developmental NOAEL: at least 15 mg/kg bw/day (based on available data from five litters)

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 2019 - 03 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the design of this study is essentially conform to the following guidelines.
• OECD Guideline 421. Reproduction/Developmental Toxicity Screening Test, July 2016.
• EPA Health Effects Test Guideline OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, July 2000.
• Council Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
• OECD Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
• EPA Health Effects Test Guideline OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
pH: 13 at concentration of 20%
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 10-11 weeks; females: 13-14 weeks
- Weight at study initiation: males: 245 - 299 g; females: 205 - 238 g
- Fasting period before study: F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before the day of scheduled necropsy, but water was available. F0- females were not fasted overnight before necropsy
- Housing: On arrival and following pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. For psychological/environmental enrichment and nesting material, animals were provided with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water ad libitum. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: at least 5 days

It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 20 - 22
- Humidity (%): 32 - 54
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 March 2019 To: 21 May 2019
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For dosing of Group 4 animals: The test item was administered as a solution as received. An adequate amount of the test item was dispensed into daily aliquots, which was stored in a controlled temperature area set to maintain 21°C until use.
For dosing of Group 2 and 3 animals: Test item dosing formulations (w/w) was homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during
dosing. Adjustment was made for specific gravity of the vehicle. A factor of 4.92 was used to correct for the purity/composition of the test item.

Dose volume: 0.0717 mL/g
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: For one couple, detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, this couple was separated 2 days after the actual mating date. The actual mating date was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For Group 4, the test item was used as received from the Sponsor; therefore, samples for dose formulation analysis for Group 4 were not collected by the Test Facility.
Samples for Analysis: Duplicate middle samples for Groups 1 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 3 (concentration and homogeneity analysis).
Dose formulation samples were collected during week 1 and week 5 of treatment.
Sample Volume: Approximately 500 mg accurately weighed into volumetric flasks of 100 mL which were filled up to mark with water.

Acceptance Criteria:
For concentration: mean sample concentration results within or equal to ±10% for solutions of target concentration.
For homogeneity: coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Duration of treatment / exposure:
Males were treated for 29 days, females that delivered were treated for 51-55 days, i.e.14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13, 14 or 15 days after delivery. Females who failed to deliver were treated for 42-43 days.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-Day dose range finding study with oral gavage of the test item.
- Rationale for animal assignment: A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
- Fasting period before blood sampling for clinical biochemistry: Males were fasted overnight, females were not fasted.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: F0-animals: Samples were collected, between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only
- How many animals: F0-animals 5/sex/group
- Parameters according to OECD 422 guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: F0-animals: Samples were collected, between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only
- How many animals: F0-animals 5/sex/group
- Parameters according to OECD 422 guideline

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood:
F0-animals: Samples were collected, between 7.00 and 10.30 a.m. on the day of scheduled necropsy
F1-animals: Samples were collected, between 7.00 and 10.30 a.m. on PND 4 at culling and PD14-16 on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only
- How many animals: F0-animals all animals, F1-animals 2 pups/litter (one male and one female, if possible)
- Parameters examined: total T4 in all F0-males and PND 14-16 pups
For the F0-generation, assessment of T4 (females) and TSH (both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 7, 8, 9, 10 and 11). These tests were performed after dosing, after completion of clinical observations.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
- Time schedule for examinations: Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
- Dose groups that were examined: all
- Parameters examined: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Sperm parameters (parental animals):
Parameters examined in male parental animals:
testis weight, epididymis weight

For the testes of all selected males of Groups 1 and 4, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups at PND 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Organ were weighed and tissues collected according to OECD 422 guideline.

HISTOPATHOLOGY: Yes
Tissues were processed and examined according to OECD 422 guideline.

Selected tissues were applicable for males that failed to sire and females that failed to deliver pups (i.e. nonmated females, non-pregnant females, no offspring): Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.

Thymus of males of Groups 2 and 3 were identified by the study pathologist during microscopic evaluation as target tissues and were evaluated and reported.

For the testes of all selected males of Groups 1 and 4, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 14-16 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations
- Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Parametric (ANOVA) tests on group means with Bonferroni correction for multiple testing) was used for the motor activity data set.
Reproductive indices:
Mating index (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100
Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Relevant clinical signs noted for the animals found dead or sacrificed in extremis are specified under Mortality.

In surviving animals, rales and/or gasping were seen incidentally in one female animal treated at 5 mg/kg bw/dayand two males and one female animal treated at 15 mg/kg bw/day. This was considered not toxicologically relevant based on the incidence observed.
Other clinical signs noted during the treatment period included alopecia, wounds and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
No findings were noted during the weekly arena observations in this study.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were four premature decedents in the study, all treated at 15 mg/kg bw/day.
One male was sacrificed after 8 days of treatment. This animal presented itself with lethargic behaviour, ventro-lateral recumbency, quick breathing and salivation. Furthermore, a bodyweight loss of 6% compared to the day before. At necropsy, this animal showed macroscopically enlarged adrenal glands with red foci, enlarged urinary bladder with red watery-clouded content, enlarged kidneys with red foci and pelvic dilation, dilated ureters, stomach with red foci and gelatinous content, gelatinous seminal vesicles and watery-clear fluid in the abdominal cavity. Microscopically, all findings were related to the presence of a large abscess in the prostate and severe inflammation and necrosis of the urinary bladder and seminal vesicles with consequently retrograde nephritis in the kidney. Most other microscopic findings were likely secondary to this inflammation (i.e. hemorrhage in adrenal glands, histiocytosis and lymphoid depletion in the marginal zone of the spleen, inflammatory cell infiltrate in the seminal vesicles and ureters, increased myeloid cells in the bone marrow). It is unclear whether these uncommon findings are (indirectly) related to the test item.

One female was found dead after 9 days of treatment. The animal was partly cannibalized and organs were severely autolytic. Therefore it was not possible to establish the cause of death.

One female was found dead after 23 days of treatment (Day 8 post-coitum). This animal presented itself with lethargic behaviour, flat and hunched posture and piloerection. Macroscopic findings of note were red-brown gelatinous contents in the jejunum and fluid in the uterus (red-brown), vagina (red-brown) and abdominal cavity (watery clear). Microscopic findings consisted of granulocytic inflammatory cell infiltrates in and around the thymic lymph node (together with aggregates of bacteria), the lung pleura and the ovarian serosa. Luminal blood was present in the uterus (corresponds to red-brown fluid in uterus/vagina). In lung and liver increased numbers of intravascular granulocytes were observed. These acute inflammatory cell infiltration at several sites together with presence of bacteria in/around the thymic lymph node, intravascular increased granulocytes and the macroscopically changes in content or fluid at several locations may indicate sepsis. This is possibly related to the gavage procedure but a relationship to the test item cannot be excluded.

One female was sacrificed after 2 days of treatment. This animal presented itself with laboured respiration, gasping and squeaking. Macroscopic findings of note were red foci in jejunum and ileum (ileum also distended with gas) and irregular surface of the glandular mucosa of the stomach. There was no clear microscopic correlate and there were no microscopic findings that could explain the moribundity of the animal.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.
During post coitum, relative food consumption was slightly lower between Day 14-20 for females treated at 15 mg/kg bw/day, reaching statistical significance between Day 14-17 post coitum. Based on the low magnitude of the change and since relative food consumption returned to control levels during the lactation phase, this was considered not toxicologically relevant.
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological and coagulation parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in haematology and coagulation parameters were considered to be unrelated to treatment given the minimal magnitude of the change and as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in functional observation parameters were noted.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and treated animals.
For males treated at 15 mg/kg bw/day, the number of total movements and ambulations were 18% and 22% lower, respectively. No statistical significance was achieved and as all values remained within the range of the historical control data, these changes were considered not to be toxicologically relevant. For females, motor activity was similar between treated and control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
There were 3/6 surviving males treated at 15 mg/kg bw/day with apoptosis in the thymus up to slight degree compared to 0/5 males in the controls. Intermediate dose groups showed incidences of 2/5 males at 1.5 mg/kg bw/day and 0/5 in males treated at 5 mg/kg bw/day. Apoptosis in the thymus can be observed as a background finding and based on the low severity and the absence of a dose relationship, the finding is considered not test item-related.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analyses: Serum levels of T4 in F0 males were considered not to be affected by treatment.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 days. An irregular cycle was noted for one female at 5 mg/kg bw/day (with normal litter). Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Description (incidence and severity):
There were 1/10 couples treated at 1.5 mg/kg bw/day, 2/10 at 5 mg/kg bw/day and 2/10 at 15 mg/kg bw/day, that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this.
Mating index was considered not to be affected by treatment. All females showed evidence of mating. The mating indices were 100% for all groups. Precoital time was considered not to be affected by treatment. All females showed evidence of mating within 4 days. Number of implantation sites was considered not to be affected by treatment.
There were 0, 1, 2 and 2 cases of non-pregnancy for the control, 1.5, 5 and 15 mg/kg bw/day groups, respectively. These numbers are within normal range. The fertility indices were 100, 90, 80 and 75% for the control, 1.5, 5 and 15 mg/kg bw/day groups, respectively. Due to the mortality of two females before start of mating, the fertility index at 15 mg/kg was slightly lower, but this was considered not toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
other: BTMAOH
Sex:
male/female
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 15 mg/kg bw/day (actual dose received)
Based on:
other: BTMAOH
Sex:
male/female
Basis for effect level:
other: Absence of adverse effects up to and including the highest dose level tested
Key result
Critical effects observed:
yes
System:
other: Mortality
Organ:
not specified
Treatment related:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
For the pups of two females (one in the 1.5 mg/kg bw/day group and one in the 5 mg/kg bw/day group) who were missing on PND 2 and PND 4, respectively, less milk in the stomach was noted on PND 1.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 100% for all groups.

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) were 100, 97, 99 and 100% for the control, 1.5, 5 and 15 mg/kg bw/day groups, respectively.
Three pups at 1.5 mg/kg bw/day and one pup at 5 mg/kg bw/day were missing on PND 2 and PND 4, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 15 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones levels:
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.
Sex ratio was considered not to be affected by treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effcets observed up to and including the highest dose level tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Results dose range finder:

A dose range finder was conducted previously to select dose levels for the main study and to determine the peak effect of occurrence of clinical signs after dosing. No testing guidelines were applicable as this dose range finder was intended for dose level selection purposes only.

A dose of 100 mg/kg bw/day was not tolerated by the animals as severe clinical signs, reduced food consumption and mild to severe body weight loss were observed. All animals treated with 100 mg/kg bw/day died before scheduled necropsy on Day 2, 5 and 6 of treatment. At dose level 25 mg/kg bw/day, some clinical symptoms, reduced food consumption and slight body weight loss were observed during the 14 Days of treatment. One animal at 25 mg/kg bw/day died at Day 8 of treatment. A dose of 15 mg/kg bw/day was well tolerated for 28 days and therefore a maximum dose level of 15 mg/kg bw/day was selected for the main study. Because of the high pH of the test item and its corrosive properties, the dose volume for the main study was set at 0.072 mL/kg, based on the dose volume used for the 15 mg/kg Group of the dose range finder. In the dose range finder, clinical signs were generally observed directly after dosing.

Dose formulation Analyses

Week 1

Accuracy

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

The concentrations analyzed in the formulations of Group 2 were in agreement with target concentrations (98% of target). The mean accuracy of the Group 3 formulation was below the target concentration (i.e. 86% of target), which was probably due to analytical issues. A new formulation analysis was scheduled in Week 5 of the toxicology study.

Week 5

Accuracy

A small response at the retention time of the test item was integrated in one of the chromatograms of the Group 1 formulation, which was assigned to noise since the peak height was within the fluctuation of the baseline. It was considered not to derive from the formulation since the response was not observed in the duplicate sample.

The concentrations analyzed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (mean accuracies 109% and 99%, respectively).

Homogeneity

The formulations of Group 2 and Group 3 were homogeneous (coefficient of variation of 2.8% and 6.1%, respectively).

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20165222) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Conclusions:
In a combined 28-day repeated dose toxicity study with reproduction/developmental toxicity test was performed according to OECD TG 422. The systemic NOAEL was 5 mg/kg bw/day as mortality was observed at 15 mg/kg bw/day. No reproductive or developmental toxicity was observed up to and including the highest dose levels and the NOAEL for reproduction and developmental was at least 15 mg/kg bw/day.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental screening toxicity test was performed according to OECD TG 422 and in accordance with GLP. Wistar rats, 10 per sex/dose were exposed to 1.5, 5 and 15 mg/kg bw/day BTMAOH. The dose levels were selected based on the dose range finder in which mortality was observed at 100 mg/kg bw/day (3/3 animals) and 25 mg/kg bw/day (1/3 animals). Because of the high pH of the test item and its corrosive properties, the dose volume for the main study was set at 0.072 mL/kg. Formulation analyses conducted during the study confirmed that formulations of test item in water were prepared accurately and homogeneously.

Parameters and endpoints evaluated in this study were according to the guideline and included: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0 -males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

 

Four early deaths were observed at 15 mg/kg bw/day: one male and one female animal were sacrificed in extremis and two female animals were found dead. The moribundity of the male animal sacrificed in extremis was most likely caused by a large abscess in the prostate and severe inflammation and necrosis of the urinary bladder and seminal vesicles with consequently retrograde nephritis in the kidney. The moribundity of the female animal sacrificed in extremis could not be explained by the macroscopic and microscopic findings. For the third animal, a female found dead, evaluation showed signs indicating sepsis. The cause of death of the other female found dead could not be established due to cannibalism and autolysis. Although there is no consistency in the morphological findings or the cause of death/moribundity of the four premature deaths and there were no similar morphological findings in the scheduled sacrificed rats, these premature death were considered test-item related given the high incidence in the highest dose group only.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical observations, body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), functional observations, macroscopic examination, organ weights, and microscopic examination).

No reproductive toxicity was observed up to the highest dose level tested (15 mg/kg bw/day).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental screening test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the design of this study is essentially conform to the following guidelines.
• OECD Guideline 421. Reproduction/Developmental Toxicity Screening Test, July 2016.
• EPA Health Effects Test Guideline OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, July 2000.
• Council Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
• OECD Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
• EPA Health Effects Test Guideline OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyltrimethylammonium hydroxide
EC Number:
202-895-5
EC Name:
Benzyltrimethylammonium hydroxide
Cas Number:
100-85-6
Molecular formula:
C10H16N.HO
IUPAC Name:
benzyltrimethylazanium hydroxide
Test material form:
liquid
Details on test material:
Appearance: Clear almost colourless liquid
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2020 (expiry date)
Specific details on test material used for the study:
pH: 13 at concentration of 20%

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 10-11 weeks; females: 13-14 weeks
- Weight at study initiation: males: 245 - 299 g; females: 205 - 238 g
- Fasting period before study: F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before the day of scheduled necropsy, but water was available. F0- females were not fasted overnight before necropsy
- Housing: On arrival and following pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. For psychological/environmental enrichment and nesting material, animals were provided with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water ad libitum. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: at least 5 days

It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 20 - 22
- Humidity (%): 32 - 54
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 March 2019 To: 21 May 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For dosing of Group 4 animals: The test item was administered as a solution as received. An adequate amount of the test item was dispensed into daily aliquots, which was stored in a controlled temperature area set to maintain 21°C until use.
For dosing of Group 2 and 3 animals: Test item dosing formulations (w/w) was homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during
dosing. Adjustment was made for specific gravity of the vehicle. A factor of 4.92 was used to correct for the purity/composition of the test item.

Dose volume: 0.0717 mL/g
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For Group 4, the test item was used as received from the Sponsor; therefore, samples for dose formulation analysis for Group 4 were not collected by the Test Facility.
Samples for Analysis: Duplicate middle samples for Groups 1 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 3 (concentration and homogeneity analysis).
Dose formulation samples were collected during week 1 and week 5 of treatment.

Acceptance Criteria:
For concentration: mean sample concentration results within or equal to ±10% for solutions of target concentration
For homogeneity: coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: For one couple, detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, this couple was separated 2 days after the actual mating date. The actual mating date was designated Day 0 post-coitum.
Duration of treatment / exposure:
Males were treated for 29 days, females that delivered were treated for 51-55 days, i.e.14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13, 14 or 15 days after delivery. Females who failed to deliver were treated for 42-43 days.
Frequency of treatment:
Once daily, 7 d/w
Duration of test:
See duration treatement / exposure
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-Day dose range finding study with oral gavage of the test item.
- Rationale for animal assignment: A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported.
- Fasting period before blood sampling for clinical biochemistry: Males were fasted overnight, females were not fasted.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 14-16
- Organs examined: Organ were weighed, tissues collected processed and examined according to OECD 422 guideline.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups at PND 13.

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

NECROPSY
- External examinations: Yes: all per litter (remaining after culling)
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Parametric (ANOVA) tests on group means with Bonferroni correction for multiple testing) was used for the motor activity data set.
Indices:
Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100
Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Relevant clinical signs noted for the animals found dead or sacrificed in extremis are specified under Mortality.

In surviving animals, rales and/or gasping were seen incidentally in one female animal treated at 5 mg/kg bw/day and two males and one female animal treated at 15 mg/kg bw/day. This was considered not toxicologically relevant based on the incidence observed.
Other clinical signs noted during the treatment period included alopecia, wounds and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. No findings were noted during the weekly arena observations in this study.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were three female premature decedents in the study, all treated at 15 mg/kg bw/day.

One female was found dead after 9 days of treatment. The animal was partly cannibalized and organs were severely autolytic. Therefore it was not possible to establish the cause of death.

One female was found dead after 23 days of treatment (Day 8 post-coitum). This animal presented itself with lethargic behaviour, flat and hunched posture and piloerection. Macroscopic findings of note were red-brown gelatinous contents in the jejunum and fluid in the uterus (red-brown), vagina (red-brown) and abdominal cavity (watery clear). Microscopic findings consisted of granulocytic inflammatory cell infiltrates in and around the thymic lymph node (together with aggregates of bacteria), the lung pleura and the ovarian serosa. Luminal blood was present in the uterus (corresponds to red-brown fluid in uterus/vagina). In lung and liver increased numbers of intravascular granulocytes were observed. These acute inflammatory cell infiltration at several sites together with presence of bacteria in/around the thymic lymph node, intravascular increased granulocytes and the macroscopically changes in content or fluid at several locations may indicate sepsis. This is possibly related to the gavage procedure but a relationship to the test item cannot be excluded.

One female was sacrificed after 2 days of treatment. This animal presented itself with laboured respiration, gasping and squeaking. Macroscopic findings of note were red foci in jejunum and ileum (ileum also distended with gas) and irregular surface of the glandular mucosa of the stomach. There was no clear microscopic correlate and there were no microscopic findings that could explain the moribundity of the animal.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. During post coitum, relative food consumption was slightly lower between Day 14-20 for females treated at 15 mg/kg bw/day, reaching statistical significance between Day 14-17 post coitum. Based on the low magnitude of the change and since relative food consumption returned to control levels during the lactation phase, this was considered not toxicologically relevant.
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological and coagulation parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in haematology and coagulation parameters were considered to be unrelated to treatment given the minimal magnitude of the change and as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in functional observation parameters were noted. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and treated animals.
For females, motor activity was similar between treated and control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in one mid dose female, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in females.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 93, 94, 97 and 95% for the control, 1.5, 5 and 15 mg/kg bw/day groups, respectively.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Gestation index was considered not to be affected by treatment. All pregnant females had live offspring. The gestation indices were 100% for all groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Duration of gestation was considered not to be affected by treatment.
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
other: BTMAOH
Basis for effect level:
mortality

Results (fetuses)

Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 100% for all groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) were 100, 97, 99 and 100% for the control, 1.5, 5 and 15 mg/kg bw/day groups, respectively.
Three pups at 1.5 mg/kg bw/day and one pup at 5 mg/kg bw/day were missing on PND 2 and PND 4, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a doserelated trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100, 100, 98 and 100% for the control, 1.5, 5 and 15 mg/kg bw/day groups, respectively.
One pup at 5 mg/kg bw/day was found dead on PND 15. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. Treatment up to 15 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 15 mg/kg bw/day (actual dose received)
Based on:
other: BTMAOH
Sex:
male/female
Basis for effect level:
other: Absence of adverse effects up to and including the highest dose level tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a combined 28-day repeated dose toxicity study with reproduction/developmental toxicity test was performed according to OECD TG 422. The systemic NOAEL was 5 mg/kg bw/day as mortality was observed at 15 mg/kg bw/day. No reproductive or developmental toxicity was observed up to and including the highest dose levels and the NOAEL for reproduction and developmental was at least 15 mg/kg bw/day.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental screening toxicity test was performed according to OECD TG 422 and in accordance with GLP. Wistar rats, 10 per sex/dose were exposed to 1.5, 5 and 15 mg/kg bw/day BTMAOH. The dose levels were selected based on the dose range finder in which mortality was observed at 100 mg/kg bw/day (3/3 animals) and 25 mg/kg bw/day (1/3 animals). Because of the high pH of the test item and its corrosive properties, the dose volume for the main study was set at 0.072 mL/kg. Formulation analyses conducted during the study confirmed that formulations of test item in water were prepared accurately and homogeneously.

Parameters and endpoints evaluated in this study were according to the guideline and included: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0 -males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

 

Four early deaths were observed at 15 mg/kg bw/day: one male and one female animal were sacrificed in extremis and two female animals were found dead. The moribundity of the male animal sacrificed in extremis was most likely caused by a large abscess in the prostate and severe inflammation and necrosis of the urinary bladder and seminal vesicles with consequently retrograde nephritis in the kidney. The moribundity of the female animal sacrificed in extremis could not be explained by the macroscopic and microscopic findings. For the third animal, a female found dead, evaluation showed signs indicating sepsis. The cause of death of the other female found dead could not be established due to cannibalism and autolysis. Although there is no consistency in the morphological findings or the cause of death/moribundity of the four premature deaths and there were no similar morphological findings in the scheduled sacrificed rats, these premature death were considered test-item related given the high incidence in the highest dose group only.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical observations, body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), functional observations, macroscopic examination, organ weights, and microscopic examination).

No reproductive toxicity was observed up to the highest dose level tested (15 mg/kg bw/day).