Reaction mass of tetramethylammonium hypochlorite and tetramethylammonium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 8, 2014 - November 19, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is considered reliable without restriction since the study is conducted according to OECD guideline 422 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Not applicable

EC Number:

940-936-5

Molecular formula:

C4H12NCl.C4H12NOCl

IUPAC Name:

Not applicable

Details on test material:

- Name of test material (as cited in study report): Halo Salt
- Physical state: liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): Al, Ca,Cr,Co,Cu,Fe,Pb,Mg,Ni,K,Na,Sn,Ti,Zn < 100 ppb
- Composition of test material, percentage of components:Tetramethylammonium Hydroxide 1.34 (wt.%), Available Chlorine 8.31 (wt.%), Stabilizer 0.12 (wt%)
- Purity test date: August 27, 2013
- Lot/batch No.: 0000054875
- Expiration date of the lot/batch: 12 August 2018
- Stability under test conditions: The test item is considered to be stable under the storage conditions provided by the Sponsor.
- Storage condition of test material: Stored ≤ 25ºC and protected from light.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: (P) approximately 64 days old
- Weight at study initiation: (P) Males: 300-500 g; Females:200-300 g
- Fasting period before study: males are fasted prior to blood collection for clinical chemistry
- Housing: All males will be individually housed in clean suspended wire mesh cages in an environmentally controlled room during acclimation and continuing until euthanasia. All females will be individually housed in clean suspended wire mesh cages in an environmentally controlled room during acclimation and continuing until mating. The cages will be elevated above cage-board or other suitable material, which will be changed at least three times each week. During cohabitation, the females will be paired (1:1) with a male from the same treatment group in a suspended wire-mesh cage (home cage of the male). Following successful mating, the females will be housed individually in a plastic cage containing ground corncob bedding material (Bed O'Cobs®) and will remain in these cages until euthanasia on lactation day 4. Nylabone® (or a similar enrichment device) will be provided to each animal
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 will be offered ad libitum during the study except during fasting of males prior to blood collection for clinical chemistry
- Water (e.g. ad libitum): ad libitum
- Acclimation period: a minimum of 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8-Jul-2014 (animal receipt) To: 31-Aug-2014 (last lactation day 4 necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing formulations were prepared at the test item concentrations indicated in the following table:
Group Number Treatment Dosage Level (mg/kg/day) Test Item Concentration (a (mg/mL) pH (b
1 Vehicle Control 0 0 5.62
2 Halo Salt 25 2.5 9.74
3 Halo Salt 50 5 10.28
4 Halo Salt 100 10 10.94
a) = The dosing formulations were not adjusted for purity.
b) = pH measurement of the first dosing formulations using a pH meter.


DIET PREPARATION
- Rate of preparation of diet (frequency): The test item formulations were prepared daily for the first 6 days of dose administration and then approximately weekly thereafter.
- Storage temperature of food: at ≤ 25ºC

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solubility and stability of Halo Salt dose formulations was established in study WIL-135506 (Hoobler, D.J., 2014). The dose formulations were analyzed for concentration using a fully validated method at WIL Research (Hoobler, D.J, 2014).

Two sets of duplicate 1.0 mL samples (from the middle stratum) were taken from the prepared dose formulations including the vehicle (middle only), from the first, approximate middle, and last dosing formulation during the in-life phase of the study. One set of samples were analyzed for concentration/homogeneity; the backup set of samples were stored at ≤25ºC. The analytical method was based on iodometric titrations.

The analyzed formulations used for dose administration met the WIL Research SOP requirement for available chlorine concentration acceptability for solution formulations, i.e., the analyzed available chlorine concentration was 90% to 110% of the theoretical chlorine concentration. No available chlorine was detected in the analyzed vehicle administered to the control group.

Duration of treatment / exposure:

Males: At least 14 days prior to mating and continuing throughout mating for a minimum of 28 days. Total of 29 doses.
Females: For 14 days prior to mating, throughout mating and continuing until one day prior to termination (lactation day 4 for females that deliver, post-mating day 25 or post-cohabitation day 25 for females that do not deliver). Total of 39-45 doses.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
25 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 5

Remarks:

Doses / Concentrations:

Basis:
other: Corresponding to 0, 2.1, 4.2, 8.3 mg available Cl2/kg bw/day

No. of animals per sex per dose:

10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Following a single oral (gavage) dose of Halo Salt at 300 mg/kg in rats, deaths (one male and one female) were noted approximately 4 hours post dose, with no abnormal physical signs in the survivors (DiDonato, 2009a).

In the most recent dose range finding study Halo Salt (0, 50, 100, and 200 mg/kg) was administered once daily via oral (gavage) for 10 days (Baracani, 2014). The study showed that at a dosage level of 200 mg/kg/day was not well tolerated in males and females as evidenced by clinical findings (tremors, ataxia, cool body and extremities, partially closed eyes, and red material around the mouth and nose), and limited body weight loss and reduced food consumption data. As a result, all animals in the 200 mg/kg/day group were found dead or euthanized in extremis by study day 4.

Lower body weights, body weight gains, and/or reduced food consumption were also noted for males at 50 and 100 mg/kg/day and females at 100 mg/kg/day. There were no effects on survival, clinical condition, macroscopic findings, or organ weights for males and females at 50 and 100 mg/kg/day or on body weights, body weight gains, and food consumption for females at 50 mg/kg/day. Based on these results, dosage levels of 0, 25, 50, 100 mg/kg bw/day were selected for this study.

Positive control:

Not needed

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males: weekly, also on the treatment days on which the FOB and Locomotor activity measurements are recorded
Females: Individual body weights and food consumption were recorded weekly, beginning one week prior to test item administration, on the first day of dosing and weekly thereafter until evidence of copulation was observed or until euthanasia (for females without evidence of mating). Following evidence of copulation, body weights and food consumption were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 29 for males and lactation day 4 for females
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males) / No (females)
- How many animals: 5 F0 animals/sex/group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study day 29 for males and lactation day 4 for females
- Animals fasted: Yes (males) / No (females)
- How many animals: 5 F0 animals/sex/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: study day 29 for males and lactation day 4 for females
- Dose groups that were examined: 5 F0 animals/sex/group
- Battery of functions tested:

Sensory observations:
Approach response
Startle response
Pupil response
Forelimb extension
Air righting reflex
Touch response
Tail pinch response
Eyeblink response
Hindlimb extension
Olfactory orientation

Neuromuscular observations:
Hindlimb extensor strength
Hindlimb foot splay
Grip strength hind and forelimb
Rotarod performance


OTHER: Anatomic pathology, histopathology

Sacrifice and pathology:

SACRIFICE
Euthanasia of adult animals were conducted by carbon dioxide inhalation.
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without examination.

GROSS PATHOLOGY: Yes (see table B Macroscopic Findings)
HISTOPATHOLOGY: Yes (see table C Microscopic Findings)

Statistics:

STATISTICAL ANALYSES
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Statistical analyses were not conducted on F0 weekly female body weight data after 1 or more animals had entered the gestation phase. Where applicable, the litter was used as the experimental unit.

ANALYSES CONDUCTED BY WIL RESEARCH
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, pre coital intervals, gestation length, numbers of former implantation sites, corpora lutea, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, and FOB data values were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. Histopathological and FOB parameters that yield scalar or descriptive data in the test item-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980). Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

Mortality:

mortality observed, treatment-related

Description (incidence):

(See section "Details on results".)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

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):

(See section "Details on results".)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

(See section "Details on results".)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results".)

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

(See section "Details on results".)

Details on results:

CLINICAL SIGNS AND MORTALITY (Table D)
One F0 male and 2 F0 females in the 100 mg/kg/day group were found dead during the study. The male animal was noted with a hindlimb caught in the wire-mesh cage flooring on the day of death, and therefore this death was not considered test item-related. One female in this group was found dead on lactation day 0; based on the proximity to parturition, this death was not considered test item related. One female in the 100 mg/kg/day group was found dead on gestation day 21; based on the presence of adverse clinical findings (cool body, partially closed eyes, salivation, and wet clear material around the mouth), this death was considered test item-related.
Test item-related clinical findings including clear or red material around the nose, eyes, and/or mouth, hypoactivity, cool body, partial or complete closure of the eye(s), tremors, and/or salivation were noted for the female that died and the majority of F0 males and females in the 100 mg/kg/day group prior to dosing and/or approximately 3 hours following dose administration intermittently throughout the study. In the 25 and 50 mg/kg/day group F0 males and females, test item-related salivation and red and/or clear material around the mouth were noted in a dose-responsive manner prior to dosing and/or approximately 3 hours following dose administration as early as study day 10; the limited occurrences at 25 mg/kg/day and at 50 mg/kg/day were not considered adverse. Limited occurrences of test item-related clinical findings persisted to the weekly examinations at all dosage levels.

BODY WEIGHT AND WEIGHT GAIN (Table E)
In the 100 mg/kg/day group F0 males, test item-related mean body weight loss or lower mean body weight gain and corresponding lower mean food consumption were noted during the pre-mating period (study days 0-13) and when the entire treatment period (study days 0-28) were evaluated. As a result, mean body weights in this group were lower (14.0% to 16.7%) than the control group throughout the entire treatment period (study days 7-28).
Test item-related slightly lower mean body weight gains and reduced food consumption were noted in the 100 mg/kg/day group F0 females during the pre mating period (study days 0-13) and continuing throughout the gestation treatment period (gestation days 0 20). As a result, mean body weights in this group were slightly lower (up to 5.6%) than the control group on gestation day 20. During lactation days 1-4, a higher mean body weight gain was noted in the 100 mg/kg/day group F0 females compared to the control group. Mean body weights in this group remained slightly lower (up to 8.3%) than the control group on lactation day 1 and 4. The slightly lower mean body weights and body weight gains noted in the 100 mg/kg/day group F0 females during the pre mating, gestation, and lactation periods were considered test item related but nonadverse. Mean body weights, body weight gains, and food consumption for F0 males and females in the 25 and 50 mg/kg/day groups were unaffected by test item administration throughout the study.

FOOD CONSUMPTION (Table F)
Test item-related slightly lower mean body weight gains and reduced food consumption were noted in the 100 mg/kg/day group F0 females during the pre mating period (study days 0-13) and continuing throughout the gestation treatment period (gestation days 0 20). As a result, mean body weights in this group were slightly lower (up to 5.6%) than the control group on gestation day 20. During lactation days 1-4, a higher mean body weight gain was noted in the 100 mg/kg/day group F0 females compared to the control group. Mean body weights in this group remained slightly lower (up to 8.3%) than the control group on lactation day 1 and 4. The slightly lower mean body weights and body weight gains noted in the 100 mg/kg/day group F0 females during the pre mating, gestation, and lactation periods were considered test item related but nonadverse. Mean body weights, body weight gains, and food consumption for F0 males and females in the 25 and 50 mg/kg/day groups were unaffected by test item administration throughout the study

HAEMATOLOGY (Table I)
There were no test item-related changes in hematology and coagulation parameters at any dosage level.

CLINICAL CHEMISTRY (Table J)
Test item-related changes in serum chemistry included higher bile acid values in the 100 mg/kg/day group F0 males, higher alanine aminotransferase values in the 50 and 100 mg/kg/day group F0 males, and higher triglyceride values in the 100 mg/kg/day group F0 females.

NEUROBEHAVIOUR (Table G)
No test item-related effects were noted

ORGAN WEIGHTS (Table K)
Suspected test item-related lower thymus and spleen weights were noted in the 100 mg/kg/day group F0 females. Higher liver weight relative to brain weights were noted in the 50 and 100 mg/kg/day group F0 females and were associated with higher absolute liver weights. Lower thymus weights (absolute and relative to body and brain weights) were noted in the 100 mg/kg/day group F0 females.

GROSS PATHOLOGY (Table B)
Test item-related small thymus was noted in the 100 mg/kg/day group females. No observations of small thymus were recorded for the males in any group.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table B)
In the 100 mg/kg/day group females the lower thymus weights correlated with a histologic finding of lymphoid depletion. Suspected test item-related microscopic findings were noted in the thymus and axillary and mesenteric lymph nodes of the 100 mg/kg/day group females. T-cell regions of the cortex of the thymus and lymph nodes were primarily affected, but in some cases, a more generalized loss of lymphocytes affecting both T-cell and B-cell regions was present.

HISTOPATHOLOGY: NEOPLASTIC (Table B)
No test item-related neoplastic changes were noted

HISTORICAL CONTROL DATA
Data obtained were compared to the WIL Research historical control data in the following endpoints studied: FOB, Motor Activity, Hematology, Serum chemistry, Organ weights

OTHER FINDINGS
FOB Assessments (Table G): No test item-related effects were noted
Locomotor Activity (Table H): No test item-related effects were noted

Effect levels

Dose descriptor:

NOAEL

Remarks:

(Systemic)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

THE TISSUES COLLECTED FOR MICROSCOPIC EXAMINATION AND WEIGHED

At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted).Microscopic examination was performed on all tissues listed below from all animals in the control and 100 mg/kg/day groups at the scheduled necropsies and all animals that were found dead. Target tissues (thymus, axillary lymph nodes, and mesenteric lymph nodes) were examined from all F₀ females. 

Adrenal glands (2)	Ovaries and oviducts (2)
Aorta	Pancreas
Bone with marrow (sternebrae)	Peripheral nerve (sciatic)
Brain^a	Pituitary gland
Coagulating glands (2)	Prostate gland
Eyes with optic nerve (2)^b	Salivary gland [mandibular (2)]
Gastrointestinal tract	Seminal vesicles (2)
Esophagus	Skeletal muscle (rectus femoris)
Stomach	Skin with mammary gland^c
Duodenum	Spinal cord (cervical)
Jejunum	Spleen
Ileum	Testes with epididymides^d(1)
Cecum	and vas deferens
	Thymus gland
Rectum	Thyroids [with parathyroids,
Heart	if present (2)]
Kidneys (2)	Trachea
Liver (sections of 2 lobes)	Urinary bladder
Lungs (including bronchi, fixed	Uterus^ewith cervix and vagina
by inflation with fixative)	All gross lesions (all groups)
Lymph node
Axillary (2)
Mandibular (2)
Mesenteric

 

                  ^a-   Histologic trimming of the brain was performed per WIL SOP such that 7 cross (coronal) sections of the brain were taken.

                  ^b      =   Placed in Davidson’s solution.

                  ^c= For females; a corresponding section of skin was taken from the same anatomic area for males.

                  ^d= Fixed in modified Davidson’s solution. 

                  ^e=  Any uterus that was placed in 10% ammonium sulfide solution for detection of implantation sites was discarded and not preserved in 10% neutral-buffered formalin.

 

The following organs were weighed from all F₀animals at the scheduled necropsies:

Adrenal glands	Ovaries with oviducts
Brain	Spleen
Epididymides^a	Testes^a
Heart	Thymus gland
Kidneys	Thyroids with parathyroids^b
Liver

                          ^a=  These paired organs were weighed separately.

                          ^b= Fixed in 10% neutral-buffered formalin prior to weighing.

Except as noted, paired organs were weighed together.

Applicant's summary and conclusion

Conclusions:

The aim was to evaluate the potential toxic effects of Halo Salt when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.
Based on mortality, adverse clinical findings, body weight deficits and corresponding reduced food consumption, serum chemistry alterations, lower organ weights, and macroscopic and microscopic findings (lymphoid depletion of the thymus) at 100 mg/kg/day, the NOAEL for F0 systemic toxicity was considered to be 50 mg/kg/day.
In addition, the following NOAELs were set up: NOAEL toxicity to reproduction 100 mg/kg bw/day, and NOAEL neonatal toxicity 50 mg/kg bw/day.

Executive summary:

The test substance was administered by daily oral gavage to male and female rats at dose levels 0, 25, 50 and 100 mg/kg bw/day (corresponding to 0, 2.1, 4.2 and 8.3 mg available Cl /kg/day). Males were exposed for 2 weeks prior to mating, during mating and up to termination (for 29 days). The females were exposed prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -45 days). Animals were observed daily for clinical signs and mortality. Furthermore, body weights and food consumption were recorded. Animals were evaluated in functional observational battery and locomotor activity and clinical pathology assessments were performed. A complete necropsy was conducted on all F0 parental animals dying spontaneously, euthanized in extremis (by carbon dioxide inhalation) or at scheduled termination.

Test item-related F0 mortality was limited to 1 female in the 100 mg/kg/day group that was found dead on gestation day 21; based on the presence of adverse clinical findings, this death was considered test item-related. There were no other test item-related deaths.

Test item-related clinical findings including clear or red material around the nose, eyes, and/or mouth, hypoactivity, cool body, partial or complete closure of the eye(s), tremors, and/or salivation were noted for the female that died and the majority of F0 males and females in the 100 mg/kg/day group prior to dosing and/or approximately 3 hours following dose administration intermittently throughout the study. In the 25 and 50 mg/kg/day group F0 males and females, test item-related salivation and red and/or clear material around the mouth were noted in a dose-responsive manner prior to dosing and/or approximately 3 hours following dose administration as early as study day 10; the limited occurrences at 25 and 50 mg/kg/day were not considered adverse in the absence of other signs of toxicity. Limited occurrences of test item-related clinical findings persisted to the weekly examinations at all dosage levels.

In the 100 mg/kg/day group F0 males, test item-related, statistically significant mean body weight loss or lower mean body weight gain and corresponding lower mean food consumption was noted during the pre-mating period (study days 0-13) and when the entire treatment period (study days 0-28) was evaluated. As a result, mean body weights in this group were statistically significantly lower (14.0% to 16.7%) than the control group throughout the entire treatment period (study days 7-28).

Test item-related slightly lower (not statistically significant) mean body weight gains and reduced food consumption were noted in the 100 mg/kg/day group F0 females during the pre‑mating period (study days 0-13) and continued throughout the gestation treatment period (gestation days 0-20). As a result, mean body weights in this group were slightly lower (up to 5.6%) than the control group on gestation day 20; differences were not statistically significant. During lactation day 1-4, a higher mean body weight gain was noted in the 100 mg/kg/day group F0 females compared to the control group; the difference was not statistically significant. Mean body weights in this group remained slightly lower (up to 8.3%) than the control group on lactation day 1 and 4; differences were not statistically significant. The aforementioned slightly lower mean body weights and body weight gains noted in the 100 mg/kg/day group F0 females during the pre‑mating, gestation, and lactation periods were considered test item‑related but nonadverse. Mean body weights, body weight gains, and food consumption for F0 males and females in the 25 and 50 mg/kg/day groups were unaffected by test item administration throughout the study.

Test item-related higher bile acid values were noted in the 100 mg/kg/day group and higher alanine aminotransferase (ALT) values were noted in the 50 mg/kg/day (+ 18.4%) and 100 mg/kg/day (+ 31.6%) groups than the control group value. Higher triglyceride values were noted in the 100 mg/kg/day group.

Test item-related small thymus was noted in 6 of 7 females in the 100 mg/kg/day group and 0 of 9 in the 0 mg/kg/day group females at the scheduled necropsy and correlated with a histologic finding of lymphoid depletion. No observations of small thymus were recorded for the males in any group.

Suspected test item-related lower thymus and spleen weights were noted in the 100 mg/kg/day group females. Higher liver weight relative to brain weight was noted in females of the 50 and 100 mg/kg/day groups and were associated with higher absolute liver weights that did not reach statistical significance.

Lower thymus weight (absolute and relative to body and brain weights) was noted in the 100 mg/kg/day group females.

Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation and are presented below

Correlations of Selected Observations

Necropsy: Thymus - small

Organ Weight: Lower thymus weight

Clinical Pathology: No correlate

Histopathology: Lymphoid depletion

Lymphoid depletion of the thymus and occasionally mesenteric and axillary lymph nodes was suspected to be related to test article administration due to the dose relationship, however, stress of pregnancy could not be ruled out as it was only seen in females (thymus) or predominantly in females (lymph nodes).

The cause of higher bile acids in the males was unclear. Although potentially an indicator of hepatobiliary damage, the lack of other significant changes in liver parameters and lack of histologic correlates or increased liver weight suggest that it may be the result of an alternate route of excretion of the halo salt.

Neonatal toxicity was evidenced at 100 mg/kg/day by lower mean numbers of pups born, live litter size, and postnatal survival; the difference from the control group was statistically significant for live litter size. Correspondingly higher numbers of pups were found dead or missing in the 100 mg/kg/day group compared to the control group. Mean F1 pup body weights (male and female) in the 100 mg/kg/day group were up to 23.2% lower than the control group.

The study was considered reliable without restriction since the study was conducted according to the current guideline (OECD422) and in compliance with GLP.