Cetrimonium bromide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The objectives of this OECD TG 422, Combined Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test were to determine the potential toxic
effects of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF when given orally by
gavage for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect
male and female reproductive performance such as gonadal function, mating behavior,
conception, parturition and early postnatal development.
In addition, parental, reproduction (up to and including implantation) and developmental
(from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
The dose levels in this study were selected to be 0, 5, 15 and 50 mg/kg/day, based on the
results of the Dose Range Finder (Test Facility Reference No. 20212914, see Appendix 6,
Appendix 7 and Appendix 8). In this Dose Range Finding Study, severe adverse local effects
in the stomach were seen at dose levels above 50 mg/kg/day.

The following parameters and end points were evaluated in this study: 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).

No adverse parental effects were recorded up to the highest dose level tested (50 mg/kg/day).
At 50 mg/kg/day, a non-adverse lower grip strength of the hindlimbs was recorded for males.
Forelimb grip strength, however, was considered unaffected by treatment with the test item.
Also, a non-adverse lower motor activity was recorded for females at 50 mg/kg/day (for
ambulations, i.e. relocation of the entire body position like walking). However, total
movements (all movements made by the animals, including ambulations but also smaller or
more fine movements like grooming, weaving or movements of the head) were considered
not affected by treatment with the test item.
Also, motor activity habituation pattern appeared similar to the concurrent control group.
These changes in grip strength and motor activity were recorded in opposite sexes and were
not associated with concurrent clinical signs or histopathological changes in examined muscle
and neuronal tissues. It was therefore considered that these variations did not represent an
adverse effect on neurobehavior of these animals. Motor activity and grip strength at 5 and
15 mg/kg/day were considered not to be affected by treatment with the test item.
Non-adverse but treatment-related changes in body weight and food intake were recorded at
50 mg/kg/day. A lower mean body weight (gain) was recorded for males (throughout
treatment) and females (post-coitum) This was accompanied by a slightly lower food
consumption during Weeks 1 and 2 of treatment (males and females), and post-coitum
(females). Body weights and food consumption at 5 and 15 mg/kg/day were considered not
affected by treatment with the test item. Other treatment-related in-life findings were confined
to piloerection recorded for all females at 50 mg/kg/day on Day 1 of treatment, and
occasionally also during Weeks 4 to 6 of the study. Given the slight magnitude of these
findings, these were considered not to represent an adverse effect of the test item.
Non-adverse but treatment-related changes in clinical biochemistry parameters at
50 mg/kg/day consisted of slightly higher alanine and aspartate aminotransferase activities
(males and females), and inorganic phosphate (males) and calcium concentration (females).
There was no apparent correlation between higher alanine and aspartate aminotransferase
activities recorded at 50 mg/kg/day and higher liver weights recorded for females at 50
mg/kg/day. These enzyme activity increases were slightly more pronounced in the opposite
sex, i.e. males, for which liver weights were similar to the control mean. Overall, the higher
liver weight for females at 50 mg/kg/day was considered not related to treatment with the test
item in absence of a clear dose-related trend and microscopic correlates. Clinical
biochemistry parameters at 5 and 15 mg/kg/day were considered not to be affected by
treatment with the test item.
Test item-related increased apoptosis of lymphocytes (mainly cortical) of the thymus was
recorded in males at 50 mg/kg/day. As there was no macroscopic correlate or related organ
weight change and at the recorded low degree, this finding was regarded non-adverse.
No local stomach effect were recorded in the Main study up to the highest dose level
administered (50 mg/kg/day). In the Dose Range Finding study, local stomach effects were
recorded at dose levels higher than the maximum dose level administered in the Main study
(i.e. at 100 mg/kg/day and higher).
No test item-related mortality occurred. One control male was sacrificed for ethical reasons
on Day 9 of treatment due to a broken upper tooth. One female at 15 mg/kg/day was
sacrificed in extremis on Day 10 of treatment showing clinical signs of ill health due to a
gavage procedure-related incident. None of these deaths were attributed to treatment with the
test item.

No treatment-related changes were noted in any of the other parameters investigated in this
study (i.e. functional observations (hearing ability, pupillary reflex and static righting reflex),
hematology and coagulation parameters, male total T4 thyroid hormone levels and
macroscopic examination).
Reproductive and developmental effects
No treatment-related changes were noted in any of the reproductive parameters investigated
in this study up to the highest dose level tested (50 mg/kg/day). (i.e. mating and fertility
indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and
histopathological examination of reproductive organs).
No treatment-related changes were noted in any of the developmental parameters investigated
in this study up to the highest dose level tested (50 mg/kg/day) (i.e. gestation, viability and
lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and
early postnatal pup development consisting of mortality, clinical signs, body weight,
anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic
examination).
At 15 mg/kg/day, developmental data was available for only seven females as two females
were not pregnant and one female was sacrificed in extremis on Day 10 of treatment. Based
on available developmental data, an adequate assessment of the study data could however still
be made.
In conclusion, test item-related parental effects at 50 mg/kg/day consisted of lower grip
strength and motor activity, lower mean body weight (gain) and food consumption,
occurrence of piloerection, changes in clinical biochemistry parameters, higher liver weights
and increased apoptosis of lymphocytes of the thymus. None of these effects were considered
adverse. Therefore, based on the results of this combined 28-day repeated dose toxicity study
with the reproduction/developmental toxicity screening test, the following No Observed
Adverse Effect Level (NOAEL) of FeF Cetyl Trimethyl Ammonium Bromide (CTAB)
USP/NF were established:
Parental NOAEL: 50 mg/kg/day
Reproduction NOAEL: 50 mg/kg/day
Developmental NOAEL: 50 mg/kg/day
Dose levels higher than 50 mg/kg/day were not tolerated based on the results of the Dose
Range Finding Study (see Appendix 6 and Appendix 7). In this Dose Range Finding Study,
all animals were sacrificed at 100 mg/kg/day. Cause of moribundity was considered to be
related to test item-related forestomach lesions in all rats consisting of a macroscopic
irregular surface with the microscopic correlate ulceration/erosion of the forestomach up to
marked degree. This was accompanied by (sub) mucosal edema (up to moderate) and
lymphogranulocytic inflammation (up to marked) and/or squamous cell hyperplasia
(minimal). Therefore, based on the severity of lesions recorded in the Dose Range Finding
Study, the highest dose level of 50 mg/kg/day was selected for the Main study for ethical
reasons. In addition, a meaningful interpretation of the study data of the Main study would
have been compromised by any severe local irritating effects that were likely to have resulted
from selecting a dose level above 50 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

NA

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

other: OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

NA

Specific details on test material used for the study:

Identification: FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF
Batch (Lot) Number: GX0B433
Expiry date: 31 December 2022 (expiry date)
Physical Description: White powder (determined by Charles River DenBosch)
Purity/Composition: 100% according to Certificate of Analysis
Storage Conditions: At room temperature

Test Facility Test Item Number: 210499
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 200°C
Chemical name (IUPAC, synonym or trade name): N,N,N-trimethylhexadecan-1-aminium bromide
CAS number: 57-09-0
EC number: 200-311-3
Molecular weight: 364.4475
Irritant or corrosive: Yes
pH: 4-6 at concentration of 100 g/L
Specific gravity / density: Not available
Solubility in vehicle: Water: Water 55 g/L (20°C)
Stability in vehicle water: Stability for at least 24 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL(Test Facility Study No. 20212913).Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed at aconcentration of 0.25 mg/mL(Test Facility Study No. 20212915).

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat strain (Crl:WI(Han) rats) from Charles River Deutschland, Sulzfeld, Germany.

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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

On arrival and following the pretest (females only) and 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, females were housed in Macrolon plastic cages (MIII type, height
18 cm). 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 left without their dam or a bottle filled
with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp
polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without
cage-enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne
GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in
which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study
No., group, animal number(s), and sex.

Environmental conditions:

Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were
maintained. The actual daily mean temperature during the study period was 19 to 24°C with
an actual daily mean relative humidity of 49 to 74%. The values that were outside the targeted
range occurred for 9 days with a maximum of 74% and were without a noticeable effect on
the clinical condition of the animals or on the outcome of the study. A 12-hour light/12-hour
dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air
recirculation) were maintained in the animal rooms.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Stock and Spiking Solutions
Stock and spiking solutions of the test item were prepared in methanol at concentrations of
1000 and 2000 mg/L.
Calibration Solutions
Six working solutions in the concentration range of 40 - 120 µg/L were prepared in methanol
from two stock solutions.
Quality Control (QC) Samples
Approximately 500 mg blank vehicle was spiked with the test item at a target concentration
of 0.25 or 2.5 mg/mL. The QC samples were treated similarly as the study samples

Details on mating procedure:

Daily, after a minimum of 14 days of treatment. The
mating period will consist of a maximum of 14
consecutive days.
Animals will be cohabitated on a 1:1 basis within the
same treatment group, avoiding sibling mating.
Detection of mating will be confirmed by evidence of
sperm in the vaginal lavage or by the appearance of an
intravaginal copulatory plug.
This day will be designated Day 0 post-coitum. Once
mating has occurred, the males and females will be
separated. A maximum of 14 days will be allowed for
mating, after which females who have not shown
evidence of mating will be separated from their males.
In case less than 9 females per group have shown
evidence of mating, each non-mated female may be remated once with a male for a maximum of 7 days (if
possible). A male of the same group having previously
shown evidence of mating

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for dose formulation analysis were not collected during the dose range finder, as
concentration, homogeneity, and stability analysis was not performed. However, to limit the
impact, the test item preparation was performed with approved procedures and documented in
detail. Preparations were visually inspected for homogeneity prior to use and all preparations
were used within 6 hours after preparation of the formulation.
Homogeneity and stability of the test item under test conditions was demonstrated in the
analytical method development and validation study (Test Facility Reference No. 20212913).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral
gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and
including the day before scheduled necropsy. This included a minimum of 14 days prior to
mating and during the mating period. Females that delivered were treated for 50-61 days, i.e.
14 days prior to mating (with the objective to cover at least two complete estrous cycles), the
variable time to conception, the duration of pregnancy and at least 13 days after delivery, up
to and including the day before scheduled necropsy. Females which failed to deliver were
treated for 42-54 days.

Frequency of treatment:

The test item and vehicle were administered to the appropriate animals by once daily oral avage 7 days a week for a minimum of 28 days.

Details on study schedule:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment
group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in
the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was
designated Day 0 post-coitum. Once mating had occurred, the males and females were
separated.
A maximum of 14 days was allowed for mating, after which females who have not shown
evidence of mating were separated from their males.
Detection of mating was not confirmed in first instance for Female No. 68. Evidence of
mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in
the assessment of the vaginal lavage, which explains the continued di-estrous during the
mating in this female. The mating date of this animal was estimated at 21 days prior to the
actual delivery date. This day was designated Day 0 post-coitum.

Dose / conc.:

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

Remarks:

Vehicle: Water

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 male/10 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

A total of 40 females will be selected at randomization before initiation of the pretest phase.
Each selected female classified as not having regular estrous cycles during the pretest phase
will be 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 will continue in
the study. The supernumerary females will then be removed from the study, and their estrous
cycle results will be kept in the raw data but will not be reported.
Animals will be randomly assigned to groups. Males and females will be randomized
separately

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Twice daily throughout the study.
Animals showing pain, distress or discomfort which was
considered not transient in nature or is likely to become
more severe, were sacrificed for humane reasons based
on OECD guidance document on humane endpoints
(ENV/JM/MONO/ 2000/7). The circumstances of any
death were recorded in detail

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
At least daily from Days 1-10, at 0-15 minutes, 1 hour
(±15 minutes) and 3 hours (± 30 minutes) after dosing.


BODY WEIGHT: Yes
- Time schedule for examinations:
On Day 1 prior to dosing and on Days 4, 9, 12, 15, 18
and 22.
In order to monitor the health status, animals at 300
mg/kg/day were also weighed on Day 2 of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
At 50 mg/kg/day, mean food consumption (before correction for body weight for males and
after correction for body weight for males and females) was slightly lower than controls
during Weeks 1 and 2 of treatment (mean over mean food consumption corrected for body
weight during premating was 0.90x and 0.87x of control for males and females, respectively).
During the post-coitum phase, mean over mean food consumption corrected for body weight
was 0.89x of control, and was primarily lower in the first week and over Days 17 to 20.
Food consumption before or after correction for body weight at 5 and 15 mg/kg/day was
considered not affected by treatment with the test item.
The statistically significantly lower absolute food consumption of females at 15 mg/kg/day
over Days 1 to 4 of lactation occurred in the absence of a dose-related trend, and was
therefore considered not related to treatment with the test item.

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: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Yes, see attached study report)
Hematological parameters of treated rats were considered not to have been affected by
treatment with the test item.

CLINICAL CHEMISTRY: Yes (see attached study report)
The following statistically significant changes at 50 mg/kg/day distinguished treated from
control animals:
• Higher alanine aminotransferase (ALT) activity in males and females (1.90x and
1.58x of control for males and females, respectively)
• Higher aspartate aminotransferase (AST) activity in males and females (1.32x and
1.23x of control for males and females, respectively)
• Higher inorganic phosphate (PHOS) concentration in males (1.18x of control)
Higher calcium (CA) concentration in females (1.06x of control)
Clinical biochemistry parameters at 5 and 15 mg/kg/day were considered not to be affected by
treatment with the test item.
Thyroid hormone analyses:
Serum levels of total T4 in F0-males were considered unaffected by treatment with the test
item.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
F0-generation: 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 12-14) These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).The following tests were performed:
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 =
abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per
animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light
conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway,
USA). Total movements and ambulations were reported. Ambulations represent
movements characterized by a relocation of the entire body position like walking,
whereas total movements represent all movements made by the animals, including
ambulations but also smaller or more fine movements like grooming, weaving or
movements of the head.

IMMUNOLOGY: No


OTHER:

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by
vaginal lavage.
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.

Sperm parameters (parental animals):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed on five
selected males of the control and 50 mg/kg/day group, on premature sacrificed male No.1,
male No. 23 of whom the female was sacrificed prematurely, and on males suspected to be
infertile. The testes revealed normal progression of the spermatogenic cycle and the expected
cell associations and proportions in the various stages of spermatogenesis were present.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- No pups were found dead/missing between lactation Days 5 and 13, resulting in a
lactation index of 100% for all groups.


PARAMETERS EXAMINED
The following parameters were examined in F1 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. Particular attention was paid to the external reproductive genitals (see attachedd study report)

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):

SACRIFICE
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25-27.
Without evidence of mating: 24-26 days after the last day of the mating period.

All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted overnight.

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were
stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

GROSS NECROPSY
- Full Gross necropsy was performed (See attached study report)

HISTOPATHOLOGY / ORGAN WEIGHTS
- A full histopathological evaluation was performed and organ weights collected in accordance to OECD 422 testing guideline requirements (see attached study report). Representative samples of tissues identified were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. All tissues as defined under Histology – F0-Generation (section 4.12.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. A peer review on the histopathology data was performed by a second pathologist.

Organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

On PND 4, the surplus pups were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. 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. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
A full histopathological evaluation was perfomed and oragn weights noted in accordance to requirements in OECD 422 testing guideline (see attached study report).

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 comparison 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

Reproductive indices:

Mating index (%); Precoital time; Fertility index (%); Gestation index (%); Duration of gestation;

Offspring viability indices:

Post-implantation survival index (%); Live birth index (%); Percentage live males/females at First Litter Check (%), Viability index (%); Lactation index (%);

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg/day, all females showed piloerection on Day 1 of treatment, and three females at this dose level also showed this symptom on a few days during Weeks 4 to 6 of the study. Salivation was recorded at increased incidence among males and females at 50 mg/kg/day after dosing. This sign was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. One male at 50 mg/kg/day (No. 38) showed hunched posture and laboured respiration following dosing on Day 1. This was ascribed to regurgitation of part of the formulation, as was seen for several other animals across the dose groups, as a result of which not all animals received the intended dose volume (for details, see Section 4.8.1). One male at 5 mg/kg/day (No. 12) was found to have a small amount of red liquid inside the mouth (presumed to be blood) after dosing on Day 122 . This latter male showed no clinical signs, had normal weight gain, and showed no necropsy findings. These incidences were ascribed to the gavage procedure and not to be related to treatment with the test item. Overall, they were considered not to have adversely affected interpretation of the study results. All other clinical signs noted during the treatment period among surviving animals 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. Also, since these clinical signs did not show any apparent dose-related trend, they were considered to be unrelated to treatment with the test item. No treatment-related clinical signs were noted during weekly arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related mortality occurred. One control male was sacrificed for ethical reasons
on Day 9 of treatment due to a broken upper tooth. One female at 15 mg/kg/day was
sacrificed in extremis on Day 10 of treatment showing clinical signs of ill health due to a
gavage procedure-related incident. None of these deaths were attributed to treatment with the
test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Non-adverse but treatment-related changes in body weight and food intake were recorded at
50 mg/kg/day. A lower mean body weight (gain) was recorded for males (throughout
treatment) and females (post-coitum) This was accompanied by a slightly lower food
consumption during Weeks 1 and 2 of treatment (males and females), and post-coitum
(females). Body weights and food consumption at 5 and 15 mg/kg/day were considered not
affected by treatment with the test item. Other treatment-related in-life findings were confined
to piloerection recorded for all females at 50 mg/kg/day on Day 1 of treatment, and
occasionally also during Weeks 4 to 6 of the study. Given the slight magnitude of these
findings, these were considered not to represent an adverse effect of the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by
treatment with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes at 50 mg/kg/day distinguished treated from
control animals:
• Higher alanine aminotransferase (ALT) activity in males and females (1.90x and
1.58x of control for males and females, respectively)
• Higher aspartate aminotransferase (AST) activity in males and females (1.32x and
1.23x of control for males and females, respectively)
• Higher inorganic phosphate (PHOS) concentration in males (1.18x of control)
• Higher calcium (CA) concentration in females (1.06x of control)
Clinical biochemistry parameters at 5 and 15 mg/kg/day were considered not to be affected by
treatment with the test item.
Thyroid hormone analyses:
Serum levels of total T4 in F0-males were considered unaffected by treatment with the test
item.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg/day, a non-adverse lower grip strength of the hindlimbs was recorded for males.
Forelimb grip strength, however, was considered unaffected by treatment with the test item.
Also, a non-adverse lower motor activity was recorded for females at 50 mg/kg/day (for
ambulations, i.e. relocation of the entire body position like walking). However, total
movements (all movements made by the animals, including ambulations but also smaller or
more fine movements like grooming, weaving or movements of the head) were considered
not affected by treatment with the test item.
Also, motor activity habituation pattern appeared similar to the concurrent control group.
These changes in grip strength and motor activity were recorded in opposite sexes and were
not associated with concurrent clinical signs or histopathological changes in examined muscle
and neuronal tissues. It was therefore considered that these variations did not represent an
adverse effect on neurobehavior of these animals. Motor activity and grip strength at 5 and
15 mg/kg/day were considered not to be affected by treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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 with the test item.
All females for which estrous cycle regularity could be determined had regular cycles of 4
days. For one female at 15 mg/kg/day (No. 67), cycle regularity in the premating phase could
not be determined as this animal only had only one complete cycle (of 4 days). This female
delivered normal offspring. 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.
For Female No. 63 at 15 mg/kg/day, cycle regularity in the premating phase could not be
determined as this animal was sacrificed in extremis on Day 10 of treatment

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed on five
selected males of the control and 50 mg/kg/day group, on premature sacrificed male No.1 and
male No. 23 of whom the female was sacrificed prematurely, and on males suspected to be
infertile. The testes revealed normal progression of the spermatogenic cycle and the expected
cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was one control couple, one couple treated at 5 mg/kg/day, two couples at
25 mg/kg/day and two couples treated at 50 mg/kg/day without offspring. One couple at
25 mg/kg/day was not mated since the female was sacrificed before mating commenced.
There were no morphological findings in the reproductive organs of either sex which could
be attributed to the test item and stage aware evaluation of the testes did not show any
indication for abnormal spermatogenesis.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed on five
selected males of the control and 50 mg/kg/day group, on premature sacrificed male No.1 and
male No. 23 of whom the female was sacrificed prematurely, and on males suspected to be
infertile. The testes revealed normal progression of the spermatogenic cycle and the expected
cell associations and proportions in the various stages of spermatogenesis were present.

No treatment-related changes were noted in any of the reproductive parameters investigated in this study up to the highest dose level tested (50 mg/kg/day). (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). No treatment-related changes were noted in any of the developmental parameters investigated in this study up to the highest dose level tested (50 mg/kg/day) (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination). At 15 mg/kg/day, developmental data was available for only seven females as two females were not pregnant and one female was sacrificed in extremis on Day 10 of treatment. Based on available developmental data, an adequate assessment of the study data could however still be made.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive parameters affected

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item. One pup (No. 11) of one control group litter (No. 47) showed partial paralysis of the left hindleg from PND 7 onwards. As this was a control pup, this was not related to treatment with the test item. All pups of one litter at 50 mg/kg/day (No. 71) that survived until scheduled necropsy showed a black tail apex from PND 7 onwards, and a missing tail apex on PND 11, 13 and/or at necropsy. Since these findings were not recorded for any other pup at this dose level, this was considered not related to treatment with the test item. The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore also considered not to be related to treatment with the test item. Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of
live offspring on PND 1) was considered not to be affected by treatment with the test item.
Viability indices were 99 and 100% for the control and 5 mg/kg/day groups, respectively, and
96% for the 15 and 50 mg/kg/day groups.
One pup of the control group, three pups at 15 mg/kg/day and four pups at 50 mg/kg/day were
found dead or missing between PND 2 and 4. Pups missing were most likely cannibalized. 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:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by
treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was 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 50 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No treatment-related changes were noted in any of the developmental parameters investigated
in this study up to the highest dose level tested (50 mg/kg/day) (i.e. gestation, viability and
lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and
early postnatal pup development consisting of mortality, clinical signs, body weight,
anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic
examination).

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

clinical biochemistry

gross pathology

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

yes

No reproductive toxicity observed at the highest dose level of 50mg/kg bw/day (actual dose received).

Conclusions:

Wistar Han rats were treated with FeF Cetyl Trimethyl Ammonium Bromide (CTAB)
USP/NF by daily oral gavage at dose levels of 5, 15 and 50 mg/kg/day. The rats of the control
group received the vehicle, Water (Elix), alone.
Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29
days). Females that delivered offspring were treated for 2 weeks prior to mating, during
mating, during post-coitum, and at least 13-15 days of lactation (for 50-61 days). Females that
failed to deliver pups were treated for 42-54 days.
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect levels (NOAEL) for reproduction was evaluated to be 50 mg/kg bw/day. For developmental toxicity, NOAEL wa established at 50 mg/kg bw/day.

Executive summary:

The objectives of this OECD TG 422, Combined Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test were to determine the potential toxic
effects of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF when given orally by
gavage for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect
male and female reproductive performance such as gonadal function, mating behavior,
conception, parturition and early postnatal development.
In addition, parental, reproduction (up to and including implantation) and developmental
(from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
The dose levels in this study were selected to be 0, 5, 15 and 50 mg/kg/day, based on the
results of the Dose Range Finder (Test Facility Reference No. 20212914, see Appendix 6,
Appendix 7 and Appendix 8). In this Dose Range Finding Study, severe adverse local effects
in the stomach were seen at dose levels above 50 mg/kg/day.

The following parameters and end points were evaluated in this study: 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).

No adverse parental effects were recorded up to the highest dose level tested (50 mg/kg/day).
At 50 mg/kg/day, a non-adverse lower grip strength of the hindlimbs was recorded for males.
Forelimb grip strength, however, was considered unaffected by treatment with the test item.
Also, a non-adverse lower motor activity was recorded for females at 50 mg/kg/day (for
ambulations, i.e. relocation of the entire body position like walking). However, total
movements (all movements made by the animals, including ambulations but also smaller or
more fine movements like grooming, weaving or movements of the head) were considered
not affected by treatment with the test item.
Also, motor activity habituation pattern appeared similar to the concurrent control group.
These changes in grip strength and motor activity were recorded in opposite sexes and were
not associated with concurrent clinical signs or histopathological changes in examined muscle
and neuronal tissues. It was therefore considered that these variations did not represent an
adverse effect on neurobehavior of these animals. Motor activity and grip strength at 5 and
15 mg/kg/day were considered not to be affected by treatment with the test item.
Non-adverse but treatment-related changes in body weight and food intake were recorded at
50 mg/kg/day. A lower mean body weight (gain) was recorded for males (throughout
treatment) and females (post-coitum) This was accompanied by a slightly lower food
consumption during Weeks 1 and 2 of treatment (males and females), and post-coitum
(females). Body weights and food consumption at 5 and 15 mg/kg/day were considered not
affected by treatment with the test item. Other treatment-related in-life findings were confined
to piloerection recorded for all females at 50 mg/kg/day on Day 1 of treatment, and
occasionally also during Weeks 4 to 6 of the study. Given the slight magnitude of these
findings, these were considered not to represent an adverse effect of the test item.
Non-adverse but treatment-related changes in clinical biochemistry parameters at
50 mg/kg/day consisted of slightly higher alanine and aspartate aminotransferase activities
(males and females), and inorganic phosphate (males) and calcium concentration (females).
There was no apparent correlation between higher alanine and aspartate aminotransferase
activities recorded at 50 mg/kg/day and higher liver weights recorded for females at 50
mg/kg/day. These enzyme activity increases were slightly more pronounced in the opposite
sex, i.e. males, for which liver weights were similar to the control mean. Overall, the higher
liver weight for females at 50 mg/kg/day was considered not related to treatment with the test
item in absence of a clear dose-related trend and microscopic correlates. Clinical
biochemistry parameters at 5 and 15 mg/kg/day were considered not to be affected by
treatment with the test item.
Test item-related increased apoptosis of lymphocytes (mainly cortical) of the thymus was
recorded in males at 50 mg/kg/day. As there was no macroscopic correlate or related organ
weight change and at the recorded low degree, this finding was regarded non-adverse.
No local stomach effect were recorded in the Main study up to the highest dose level
administered (50 mg/kg/day). In the Dose Range Finding study, local stomach effects were
recorded at dose levels higher than the maximum dose level administered in the Main study
(i.e. at 100 mg/kg/day and higher).
No test item-related mortality occurred. One control male was sacrificed for ethical reasons
on Day 9 of treatment due to a broken upper tooth. One female at 15 mg/kg/day was
sacrificed in extremis on Day 10 of treatment showing clinical signs of ill health due to a
gavage procedure-related incident. None of these deaths were attributed to treatment with the
test item.

No treatment-related changes were noted in any of the other parameters investigated in this
study (i.e. functional observations (hearing ability, pupillary reflex and static righting reflex),
hematology and coagulation parameters, male total T4 thyroid hormone levels and
macroscopic examination).
Reproductive and developmental effects
No treatment-related changes were noted in any of the reproductive parameters investigated
in this study up to the highest dose level tested (50 mg/kg/day). (i.e. mating and fertility
indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and
histopathological examination of reproductive organs).
No treatment-related changes were noted in any of the developmental parameters investigated
in this study up to the highest dose level tested (50 mg/kg/day) (i.e. gestation, viability and
lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and
early postnatal pup development consisting of mortality, clinical signs, body weight,
anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic
examination).
At 15 mg/kg/day, developmental data was available for only seven females as two females
were not pregnant and one female was sacrificed in extremis on Day 10 of treatment. Based
on available developmental data, an adequate assessment of the study data could however still
be made.
In conclusion, test item-related parental effects at 50 mg/kg/day consisted of lower grip
strength and motor activity, lower mean body weight (gain) and food consumption,
occurrence of piloerection, changes in clinical biochemistry parameters, higher liver weights
and increased apoptosis of lymphocytes of the thymus. None of these effects were considered
adverse. Therefore, based on the results of this combined 28-day repeated dose toxicity study
with the reproduction/developmental toxicity screening test, the following No Observed
Adverse Effect Level (NOAEL) of FeF Cetyl Trimethyl Ammonium Bromide (CTAB)
USP/NF were established:
Parental NOAEL: 50 mg/kg/day
Reproduction NOAEL: 50 mg/kg/day
Developmental NOAEL: 50 mg/kg/day
Dose levels higher than 50 mg/kg/day were not tolerated based on the results of the Dose
Range Finding Study (see Appendix 6 and Appendix 7). In this Dose Range Finding Study,
all animals were sacrificed at 100 mg/kg/day. Cause of moribundity was considered to be
related to test item-related forestomach lesions in all rats consisting of a macroscopic
irregular surface with the microscopic correlate ulceration/erosion of the forestomach up to
marked degree. This was accompanied by (sub) mucosal edema (up to moderate) and
lymphogranulocytic inflammation (up to marked) and/or squamous cell hyperplasia
(minimal). Therefore, based on the severity of lesions recorded in the Dose Range Finding
Study, the highest dose level of 50 mg/kg/day was selected for the Main study for ethical
reasons. In addition, a meaningful interpretation of the study data of the Main study would
have been compromised by any severe local irritating effects that were likely to have resulted
from selecting a dose level above 50 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No treatment-related changes were noted in any of the developmental parameters investigated
in this study up to the highest dose level tested (50 mg/kg/day) (i.e. gestation, viability and
lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and
early postnatal pup development consisting of mortality, clinical signs, body weight,
anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic
examination).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available