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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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: The study is considered reliable without restrictions since the study is conducted according to the guideline OECD 422 and in compliance with GLP.
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
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)
Route of administration:
oral: gavage
Vehicle:
water
Details on 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

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: the appearance of a vaginal copulatory plug or by evidence of sperm in a vaginal lavage. referred to as day 0 of pregnancy
- If no evidence of copulation is obtained after 14 days, the animals will be separated without further opportunity for mating
- After successful mating each pregnant female was caged (how): housed individually in a plastic cage containing ground corncob bedding material (Bed O'Cobs®) and remained in these cages until euthanasia on lactation day 4.
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.

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. The results of the available mean chlorine concentration and percent of target values are summarized below.

Test Item Concentration in Formulations
Mean Available Chlorine Concentration, mg/mL (% of Target)
Date of Preparation Group 1* Group 2* Group 3* Group 4*
(0 mg Halo Salt /mL) (2.5 mg Halo Salt/mL) (5 mg Halo Salt/mL) (10 mg Halo Salt/mL)
17-Jul-2014 ND 7.94(95.7) 8.33 (100) 8.24 (99.3)
08-Aug-2014 ND 7.80 (94.0) 8.51 (103) 8.51 (103)
20-Aug-2014 ND --- --- ---
27-Aug-2014 --- 8.51 (103) 8.51 (103) 8.51 (103)
ND = No available chlorine detected
“---” = Not analyzed
* = The theoretical available chlorine (%) is 8.3
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
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
25 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
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 and 10 females per dose
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 < 50 mg/kg bw/day and 50 mg/kg bw/day were considered to be the no-observed-adverse-effect levels (NOAEL) for systemic toxicity for males and females, respectively.
Positive control:
Not needed
Parental animals: Observations and examinations:
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

OTHER: FOB Assessments, Locomotor Activity, Clinical Pathology, Hematology, Serum Chemistry, Anatomic Pathology
Oestrous cyclicity (parental animals):
Not reported
Sperm parameters (parental animals):
Not investigated. Only testis weights were recorded.
Litter observations:
STANDARDISATION OF LITTERS
No. On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without examination.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, gross malformations, weight gain, adverse changes in appearance and behavior

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
- Male animals: All surviving animals
A complete necropsy was conducted to all male F0 parental animals. One male animal was found dead during the study. All other males were euthanized following completion of the mating period.
- Maternal animals: All surviving animals
Females that delivered were euthanized on lactation day 4
Females that failed to deliver were euthanized on post mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating).
Females with total litter loss were euthanized within 24 hours of litter loss.

GROSS NECROPSY
- Gross necropsy consisted of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examination was performed on all tissues (see Table A) 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.
The tissues weighed were also indicated in the Table A.
Postmortem examinations (offspring):
SACRIFICE
- On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without examination. Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues of intact offspring that were found dead were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross 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).
Reproductive indices:

Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = No. of Males Siring a Litter / Total No. of Males Used for Mating x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100

Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100

Pre-Coital Interval (days)
Offspring viability indices:
Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4 / No. of Pups Born Per Litter) / No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N / Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group x 100

When N = PND 0-1 and 1-4
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(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".)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(See section "Details on results".)
Other effects:
not examined
Description (incidence and severity):
Test substance intake: The test substance was administered by oral gavage
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
(See section "Details on results".)
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) (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 were not considered adverse. Limited occurrences of test item-related clinical findings persisted to the weekly examinations at all dosage levels.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) (Table E & F)
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 non adverse. 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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) (Table M)
Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

ORGAN WEIGHTS (PARENTAL ANIMALS) (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 (PARENTAL ANIMALS) (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 (PARENTAL ANIMALS) (Table C)
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.

OTHER FINDINGS (PARENTAL ANIMALS) See Repeated dose toxicity:oral_key study (OECD 422) RSS for result tables G, H, I and J.
FOB Assessments: No test item-related effects were noted (Table G)
Locomotor Activity: No test item-related effects were noted (Table H)
Hematology: There were no test item-related changes in hematology and coagulation parameters at any dosage level. (Table I)
Serum Chemistry: 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. (Table J)
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'
Dose descriptor:
NOAEL
Remarks:
(Reproduction)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level tested
Clinical signs:
no effects observed
Description (incidence and severity):
(See section "Details on results".)
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
(See section "Details on results".)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(See section "Details on results".)
Sexual maturation:
not examined
Description (incidence and severity):
On PND 4, surviving F1 rats were euthanized
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Only pups found dead during PND 0-4 were examined. (See section "Details on results".)
Histopathological findings:
not examined
VIABILITY (OFFSPRING) (Table L)
Lower mean numbers of pups born and live litter size were noted in the 100 mg/kg/day group (13.1 and 11.1 pups, respectively) compared to the control group (14.9 and 14.8 pups, respectively); the difference was significant (p<0.05) for live litter size. In addition, the mean live litter size in this group was below the minimum mean value in the WIL Research historical control data (11.6 pups). The aforementioned differences were considered test item-related.

Postnatal survival of the pups from birth to PND 4 in the 100 mg/kg/day group (56.6% per litter) was lower than the control group (97.9% per litter) and the minimum mean value in the WIL Research historical control data (83.8% per litter). Although the difference was not statistically significant, the lower postnatal survival in the 100 mg/kg/day group was considered test item-related. These results were partially attributed to female no. 23104 in this group that was noted with 10 dead pups on PND 0. The mean number of pups born, live litter size, and postnatal survival in the 25 and 50 mg/kg/day groups and the percentage of males at birth in the 25, 50, and 100 mg/kg/day groups were similar to the control group values. Postnatal survival in the 50 mg/kg/day group from birth to PND 4 (86.3% per litter) was slightly lower than the control group. However, the difference from the control group was not statistically significant and was attributed to 1 female (no. 23112) in this group with a total litter loss, and therefore was not considered test item-related.

CLINICAL SIGNS (OFFSPRING) (Table L)
No test item-related clinical findings for F1 pups were noted at any dosage level.

BODY WEIGHT (OFFSPRING) (Table L)
Mean PND 1 F1 male and female pup body weights (5.6 g and 5.4 g, respectively) in the100 mg/kg/day group were lower (22.2% and 20.6%, respectively) than the concurrent control group values (7.2 g and 6.8 g, respectively) and the minimum mean values in the WIL Research historical control data (6.7 g and 6.4 g, respectively). The differences from the concurrent control group were significant (p<0.01). Although mean male and female pup body weight gains in the 100 mg/kg/day group were similar to the control group during PND 1-4, mean F1 male and female pup body weights remained lower (23.2% and 15.6%, respectively) on PND 4. The difference from the concurrent control group was significant (p<0.01) for F1 male pups. The aforementioned lower F1 pup body weights were considered test item-related. Mean F1 male and female pup body weights and body weight changes during PND 1-4 in the 25 and 50 mg/kg/day groups were unaffected by parental administration of the test item. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING) (Table L)
No test item-related macroscopic findings for F1 pups that were found dead were noted at any dosage level. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.
Dose descriptor:
NOAEL
Remarks:
(Development)
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on lower postnatal survival and lower mean numbers of pups born, live litter size, and F1 pup body weights.
Reproductive effects observed:
not specified
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.
Under the conditions of this screening study, no test item-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 100 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity. Based on the lower mean numbers of pups born, live litter size, and postnatal survival at 100 mg/kg bw/day, the NOAEL for neonatal toxicity was considered to be 50 mg/kg/day. However, these developmental changes seen are considered minor and non-specific secondary consequence of maternal toxicity.
In addition, the NOAEL for F0 systemic toxicity was set up at 50 mg/kg/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. Furthermore, 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 was evaluated. 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 in the following table.

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.

No test item-related effects on reproductive performance were observed at any dosage level. 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 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
Additional information

In a study by Baracani, R. (2015) potential reproduction and developmental toxicity of the target substance have been evaluated in a screening test according to OECD Guideline 422. The study is used as the key study for deriving a NOAEL for ES & RC for the oral route.

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). The oral route of administration is regarded the most likely route of human exposure. Historically, this route has been used extensively for studies of this nature. 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.

This study was designed to evaluate the potential toxic effects of Halo Salt in rats and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

A validated analytical method based on iodometric titrations for the determination of the available chlorine content in Halo Salt solution formulations was used to verify test item available chlorine concentration. The samples from test formulations were collected from the first, approximate middle and last dosing formulations prepared during the in-life phase of the study. The theoretical available chlorine (%) in Halo Salt solution formulations is 8.3. The analysed available chlorine concentration was 90% to 110% of the theoretical chlorine concentration. The test article was not detected in the vehicle formulation that was administered to the control group.

F0 generation

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 non adverse. 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 was 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.

Reproductive performance

No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item‑treated groups. Lower male fertility and copulation indices were noted in the 100 mg/kg/day group compared to the concurrent control group values. However, the lower values in this group were within WIL Research historical control data range, and therefore were not considered test item-related. One male each in the 50 and 100 mg/kg/day groups did not sire a litter. One female in the 100 mg/kg/day group had evidence of mating but did not deliver.

The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

F1 litter data

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. The observed lower mean number of pups born at 100 mg/kg/day (13.1) was not statistically significant compared to the control group (14.9). The lower live litter size at 100 mg/kg/day (11.1) was significant compared to the control group (14.8). However, this effect is considered minor change and seen in association with maternal toxicity.

Postnatal survival of the pups from birth to PND 4 in the 100 mg/kg/day group (56.6% per litter) was lower than the control group (97.9% per litter) and the minimum mean value in the WIL Research historical control data (83.8% per litter). The difference was not statistically significant and these results were partially attributed to one female in 100 mg/kg/day that was noted with 10 dead pups on PND 0.

Mean PND 1 F1 male and female pup body weights (5.6 g and 5.4 g, respectively) in the100 mg/kg/day group were lower (22.2% and 20.6%, respectively) than the concurrent control group values (7.2 g and 6.8 g, respectively) and the minimum mean values in the WIL Research historical control data (6.7 g and 6.4 g, respectively). The differences from the concurrent control group were significant (p < 0.01). Mean male and female pup body weight gains in the 100 mg/kg/day group were similar to the control group during PND 1-4. The lower body weights observed in the study are considered minor changes and seen in association with maternal toxicity.

Conclusions

Based on mortality, adverse clinical findings (hypoactivity, salivation, cool body, red and/or clear material on various body surfaces), 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.

Under the conditions of this screening study, no test item-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 100 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of Halo Salt when administered orally by gavage to Crl:CD(SD) rats.

Lower postnatal survival and lower mean numbers of pups born, live litter size, and F1 pup body weights were observed at 100 mg/kg/day. Thus, the NOAEL for neonatal toxicity derived by authors was set at 50 mg/kg/day. However, only statistically significant findings were the lower live litter size and lower pup body weights which were considered minor. Furthermore, the neonatal effects observed could be due to non-specific secondary consequence of maternal toxicity.

There are no additional data on Halo Salt itself. However, Halo Salt exists as a solution in which the composition of ions depends on the pH. Considering the biological systems, the active chemical species available are tetramethylammonium hypochlorite and tetramethylammonium chloride in equilibrium in the pH range 6-8, and Cl2 at pH below 4. As a consequence, when Halo Salt is administered orally, it is transformed to chlorine in the stomach. For this reason, the toxicological profile of Halo Salt is linked to that of chlorine.

In a one well conducted study potential reproductive effects using chlorine (as HOCl) were assessed in rats (Carlton et al., 1986 cited in EU, 2007b). The test substance was administered in solution by gavage or in drinking water. Males (12 per group) were administered doses of 0, 1, 2, and 5 mg/kg body weight of aqueous chlorine. Administration by gavage started 56 days prior to breeding and continued throughout the 10 day breeding cycle. Female rats (24 per group) received the same concentrations by gavage for 14 days prior to breeding and throughout breeding, gestation and lactation, until the pups were weaned on day 21. There were no dose-related effects on fertility, foetal viability, litter size, foetal body weight, day of eye opening or day of vaginal patency. As no severe effects were shown in all dosed groups, the value of 5 mg/kg bw available chlorine was established as NOEL for reproductive toxicity.


Short description of key information:
No test item-related effects were observed on reproductive performance at any dosage level when administered orally by gavage to Crl:CD(SD) rats. Therefore, NOAEL for reproductive performance is 100 mg/kg bw/day (the highest concentration level tested).

Based on lower postnatal survival and lower mean numbers of pups born, live litter size, and F1 pup body weights at 100 mg/kg/day, the NOAEL for neonatal toxicity was set by authors at 50 mg/kg/day. These developmental changes seen are considered minor and non-specific secondary consequence of maternal toxicity.

Halo Salt is marketed and used in the EU approximately 1:2 water diluted product (trade name CLK-222). ES & RC are conducted for this diluted formulation of Halo Salt.

Further relevant information on toxicity to reproduction is available from the study conducted for chlorine. In this study, no test substance related effects on reproduction were observed in any dosed groups.

The available studies are sufficient in their design and quality to draw the conclusion that there is no evidence to suggest that Halo Salt would present adverse effects on development or fertility.

Justification for selection of Effect on fertility via oral route:
A good quality study was conducted for the target substance according to the OECD guideline 422 and in compliance with GLP.

Justification for selection of Effect on fertility via inhalation route:
Not a likely exposure route, since the target substance has low evaporation rate from aqueous solution.

Justification for selection of Effect on fertility via dermal route:
Not a likely exposure route. As the substance is classified for skin corrosive appropriate RMMs need to be worn to prevent dermal exposure.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

See section Effects on fertility above.


Justification for selection of Effect on developmental toxicity: via oral route:
No study was selected since endpoint conclusion is based on a good quality study conducted for the target substance (OECD 422) and on a relevant reliable information available from chlorine.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Not a likely exposure route, since the target substance has low evaporation rate from aqueous solution. 

Justification for selection of Effect on developmental toxicity: via dermal route:
Not a likely exposure route. As the substance is classified for skin corrosive appropriate RMMs need to be worn to prevent dermal exposure

Justification for classification or non-classification

Based on the results of the reproduction/developmental toxicity screening test and relevant information from analogue substances there is no evidence to suggest that Halo Salt would present adverse effects on fertility or development. Therefore, the Halo salt will not be classified for reproductive toxicity in accordance with the criteria of CLP Regulation 1272/2008 and according to the EU directive 67/548/EEC.

Additional information