Cetrimonium bromide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

Dose Range Finding Study for the OECD 422 study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiated October 2019, Dose Range Finding (DRF) Study October 2019, Necropsy November 2019.

Reliability:

1 (reliable without restriction)

Principles of method if other than guideline:

DRF study for “Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening of FeF cetyl trimethyl ammonium bromide (CTAB) USP/NF by oral gavage in rats” to follow OECD Guideline 422. Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016 and EPA Health Effects Test Guideline OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

GLP compliance:

yes

Remarks:

DRF study perfomed according to GLP,. Reported in Appendix 6, Appendix 7 and Appendix 8 of the report on main study 20212915

Specific details on test material used for the study:

White powder
Batch (Lot) Number: GX0B433
Expiry date: 31 December 2022
Purity: 100% according to certificate of analysis

Species:

rat

Strain:

Wistar

Sex:

female

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose levels: 0, 50, 100, 200, 300 mg/kg bw/day

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

No. of animals per sex per dose:

3 female rats/ group

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats treated at 100, 200 and 300 mg/kg/day were sacrificed moribund at Day 5, 4 and 3 respectively. Cause of moribundity were considered test item-related forestomach lesions in all rats in the form of the macroscopic finding 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).
Furthermore, findings in the glandular stomach were noted which consisted of a focal ulcer at 300 mg/kg/day (minimal) and from 200 mg/kg/day onward a slightly increased incidence of granulocytic infiltrate (minimal). Although these findings can be seen as spontaneous background findings, a relationship with treatment with the test item for these latter two findings cannot be excluded.
Minor findings were noted for the intestines which included a minor increase in incidence and/or severity of inflammatory cell infiltrate (up to slight) at 200 and/or 300 mg/kg/day and epithelial necrosis in the duodenum of one rat treated at 300 mg/kg/day (minimal). A test item-relationship cannot be excluded.
There was no microscopic correlate for the macroscopic finding gelatinous contents (considered test item-related) noted in the stomach of all 300 mg/kg/day treated rats and in the intestinal tract of all rats treated at 100, 200 and 300 mg/kg/day.
There were no test item-related morphologic alterations in rats treated at 50 mg/kg/day for 14 consecutive days.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Remarks on result:

not determinable because of methodological limitations

Reproductive effects observed:

not specified

Conclusions:

Adverse test item-related morphologic alterations following the administration of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF were noted at 100, 200 and 300 mg/kg/day in the form of an early onset of moribundity related with marked forestomach ulceration/erosion and concomitant findings, as edema, inflammation and squamous cell hyperplasia.

Treatment for 14 days at 50 mg/kg/day was well tolerated.
Due to the observed steep dose-reponse relationship for forestomach ulceration/erosion and because of longer exposure period in the main study, the maximum dose in the main study should be no higher than 50 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

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

Test guideline 5

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

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

Test guideline 6

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

Test material

Test material information

Constituent 1

Reference substance name:

Cetrimonium bromide

EC Number:

200-311-3

EC Name:

Cetrimonium bromide

Cas Number:

57-09-0

Molecular formula:

C19H42N.Br

IUPAC Name:

cetrimonium bromide

Test material form:

solid: particulate/powder

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).

Test animals

Species:

rat

Strain:

Wistar

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.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. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Identification:

Prior to start of the pretest period (females) or treatment period (males), each animal was identified using a chip. Prior to the pretest period, reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by a chip.Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Environmental Acclimation

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7days prior to start of the pretestperiod (females) or 7days beforethe commencement of dosing (males).

Selection, Assignment, Replacement, and Disposition of Animals

A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classifiedas 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 of40 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.Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

Housing

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, malesand 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.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected becausein order to optimize the potential occurrence of systemic toxicity for the purpose of regulatory hazard assessment.
The dose levels were selected based on the results of a 14-day Dose Range Finder with oral gavage administration of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NFin rats (Test Facility ReferenceNo.20212914, seeAppendix 6), and in an attempt to produce graded responses to the test item. In this Dose Range Finding Study, all animals were sacrificed at 100 mg/kg/day. Therefore, based on ethical considerations and in order to allow a meaningful interpretation of the study data at the highest selected dose level, the highest dose level of 50 mg/kg/day was selected for the Main study.

Vehicle:

water

Details on oral exposure:

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. Dosing formulations containing vehicle only were dosed within a similar timeframe after preparation as test item dosing formulations, when practically possible.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. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

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 gavage 7 days a week for a minimum of 28 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle (water)

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10 male/10 female/per dose (0, 5, 15, 50 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

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.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

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
Animals were weighed individually on the first day of treatment (prior to dosing), 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.
A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Over Days 1-4, 4-9, 9-12, 12-15, 15-18 and 18-22.

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
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Yes, see attached study report)
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due
to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0 females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single
pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of
the necropsy procedure.

CLINICAL CHEMISTRY: Yes (see attached study report)
Blood samples at a target volume of 0.5 mL (plasma) were collected into tubes containing
Li-Heparin as anticoagulant. Serum samples at a target volume of 0.5 mL were collected in
tubes without anticoagulant.
Blood samples were processed for plasma or serum (bile acids), which was analyzed for the
parameters specified in the following:

Alanine aminotransferase (ALAT) Creatinine
Aspartate aminotransferase (ASAT) Glucose
Alkaline phosphatase (ALP) Cholesterol
Total protein Sodium
Albumin Potassium
Total bilirubin Chloride
Bile acids Calcium
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic phosphate (Inorg. Phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
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.

IMMUNOLOGY: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table in attached study report)

HISTOPATHOLOGY: Yes (see table in attached study report)

Other examinations:

Anogenital distance (absolute and normalized for body weight) in male and female pups was
considered not to be affected by treatment with the test item.

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

Results and discussion

Results of examinations

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. Clinical signs recorded for animals that were sacrificed are specified under Section 9.2.1 (Mortality).

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:

effects observed, non-treatment-related

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

Description (incidence and severity):

A finding of note was observed at 50 mg/kg/day consisting of, a statistically significant
higher absolute liver weight in females.
The increase was minor, occurred in the absence of a clear dose-related trend and had no
microscopic correlate. Therefore, this liver weight increase was considered not test-itemrelated and non-adverse.
Any other differences, including those that reached statistical significance (absolute heart
weight and relative kidney weight at 50 mg/kg/day (males), relative thymus weight at 15
mg/kg/day (males) , and absolute and relative adrenal gland weight at 5 and 50 mg/kg/day
(females)) were considered not to be Fef Cetyl Trimethyl Ammonium Bromide (CTAB)
USP/NF-related due to the lack of dose-related pattern, lack of a test item-related
microscopic correlate and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with FeF Cetyl Trimethyl Ammonium
Bromide (CTAB) USP/NF were noted in the thymus of the 50 mg/kg/day group males and
are summarized in report.
Increased apoptosis of lymphocytes (mainly cortical) of the thymus was recorded in males of
the 50 mg/kg/day group (minimal degree).
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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

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 systemic effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

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 in accordance with OECD 422. 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 systemic toxicity was evaluated to be 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).