Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined repeated dose toxicity and reproductive-developmental screening toxicity study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 04, 2020 to March 24, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose toxicity and reproductive-developmental toxicity screening study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 04, 2020 to March 24, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty-three male and 46 female Crl:WI(Han) rats were obtained from Charles River Laboratories Margate United Kingdom, in order to provide sufficient animals for study selection.

Upon arrival, males were 9 to 10 weeks old and females were 7 to 8 weeks old. At the start of dosing, males weighed between 274.8 and 380.7 g and were 11 to 12 weeks old, females weighed between 170.9 and 223.9 g and were 10 to 11 weeks old.

Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for 2 weeks prior to initiation of dosing (males) or 1 week prior to smearing (females), and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes.

Animals were housed in groups (up to four animals/cage by sex [both sexes pre-pairing and males post-pairing] or with one female and one male [pairing]), individually (mated females), or with their litter (females during lactation).

Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed by Covance for specific contaminants.

Animals had ad libitum access to SDS Rat and Mouse breeder diet VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.

Animals were provided with wooden Aspen chew blocks and rodent retreats. During gestation and lactation, nesting materials (i.e. paper wool) were provided as forms of environmental enrichment.

Upon arrival, animals were assigned to dose groups using an internal randomization procedure. Animals were individually identified by electronic implant. Animals selected for FOB assessments were identified by tail markings.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test article was administered once daily orally by gavage. Males were dosed for 42 consecutive days (2 weeks prior to pairing, 1 week during the pairing phase, and 3 weeks post-pairing until the day before necropsy). Females were dosed for up to 54 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13).

A dose volume of 10 mL/kg was used. Individual dose volumes were based on the most recent individual body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared for use in Week 1 and 6 were analyzed to determine the achieved concentration. Triplicate samples were taken from the middle of the test article formulations and were analyzed.

A single sample was taken from the middle of the control formulations and was analyzed.

Duration of treatment / exposure:

42 days for males and 54 days for females

Frequency of treatment:

Once daily orally by gavage

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 rats/ sex/ group

Control animals:

yes

Positive control:

The control article was the vehicule (purified water)

Observations and examinations performed and frequency:

HEALTH MONITORING
All animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.

CLINICAL EXAMINATIONS
Each animal was given a detailed physical examination daily (at a similar time on each day) from the first day of dosing until the day of necropsy.

POSTDOSE OBSERVATIONS
Initially, animals were observed daily prior to dosing and immediately after dosing upon to return to home cage until Pre-Pairing Day 14, inclusive. A few incidences of mouth rubbing following dosing upon return to the home cage following dosing were noted for animals administered 80 mg/kg/day and postdose opbservations were recommenced from Pairing Day 3 for males and Pairing Day 3 or GD 0/1/2 for females.

BODY WEIGHTS
Male body weights were recorded once on the day prior to dose initiation, on the first day of dosing and at weekly intervals thereafter, and on the day prior to necropsy (PostPairing Day 21). A fasted terminal body weight was recorded at necropsy on PostPairing Day 22.
Female body weights were recorded once on the day prior to dose initiation; on the first day of dosing and weekly prior to pairing; on GD 0, 7, 14, and 20; and on LD 0 (where applicable) 1, 4, 7, and 13. A fasted terminal body weight was recorded at necropsy on LD 14.

FOOD CONSUMPTION
Male food consumption was determined once weekly prior to pairing and post-pairing until the day prior to necropsy.
Female food consumption was determined once weekly prior to pairing; from GD 0 to 7, 7 to 14, and 14 to 20; and from LD 1 to 4, 4 to 7, and 7 to 13.

ESTROUS CYCLE DETERMINATION
Daily vaginal washings (lavage) were taken from all females for 14 consecutive days prior to dosing, from the start of dosing until the confirmation of mating, and on the morning of LD 14, prior to necropsy.

MATING
Animals were paired on the day following 15 days of dosing. During the pairing phase, one male was housed for up to 7 days with one female from the same dose group.

PARTURITION
Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25.

Statistics:

Analysis of variance (ANOVA) and pairwise comparisons, as applicable, were used to analyze the following.

- Body weight (adult animals)
- Body weight change (adult animals)
- Food consumption
- Thyroid hormone analysis (male and female pups), where a single sample for each pup is obtained. No analysis will be performed for pooled samples.

Prior to the performing the ANOVA, Levene’s test was performed to test for equality of variances between groups:

- Where Levene’s test was significant (P ≤ 0.05), a rank transformation (to stabilize the variances) was applied before the ANOVA was conducted (note: Levene’s test was not applied to the rank-transformed data).
- Where Levene’s test was not significant (P > 0.05), ANOVA was conducted.


For comparisons to a single control:
- If the group effect of the ANOVA was significant (P ≤ 0.05), Dunnett’s test was used for pairwise comparisons between each test article-treated and control group.
- If the ANOVA was not significant (P > 0.05), the Dunnett’s test results were not applicable to the evaluation and were not reported or used to interpret study data.

Anogenital distance was analyzed using analysis of covariance (ANCOVA). Cube root litter weight was taken as the covariate within the analysis.

Data containing values above/below the limit of quantitation were not analyzed, and the tables were footnoted accordingly. Where insufficient data were available for meaningful analysis, no analysis was performed, and the tables were footnoted accordingly.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the pre-pairing phase (Days 1 through 15), mean body weight gain for males administered 80 mg/kg/day was marginally lower than controls, but differences from controls did not attain statistical significance. Overall body weight gain in these animals was approximately 11% lower than controls; however, in view of the minimal impact on absolute body weight, no clear dose-response, and no effect on associated food consumption,. this finding was considered of no toxicological significance.

Compared with controls, a few instances of lower body weight gains were also observed for males administered 40 mg/kg/day.; However, no dose relationship was evident, and this observation was considered incidental. No effect of test article on body weight gain was evident for males administered 20 mg/kg/day or females administered ≥20 mg/kg/day, compared with controls.

Food efficiency:

no effects observed

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

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No change in hematology or coagulation was observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared with controls, noteworthy or statistically significant changes in clinical chemistry parameters included minimally increased bile acids in females administered ≥20 mg/kg/day, minimally increased cholesterol in females administered 80 mg/kg/day (P < 0.05), minimally increased calcium in males administered ≥20 mg/kg/day (P < 0.05), and minimally increased inorganic phosphorus (P < 0.01) and minimally decreased chloride in females administered 80 mg/kg/day (P < 0.05). These changes were minor, generally without dose-relationship, limited to one or the other sex, and, in the absence of any microscopic correlates, were considered of no toxicological significance.

Endocrine findings:

no effects observed

Description (incidence and severity):

No related effect on T4 or TSH was observed for either sex at any dose level.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No related thyroid weight or thyroid:body weight ratio changes were recorded for F1 pups from litters of F0 animals administered 20, 40, or 80 mg/kg/day.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared with controls, statistically significantly increased basic and fine movements were observed during the first 5 or 10-minutes of assessment (females and males, respectively) at 80 mg/kg/day (P < 0.05). Statistically significant increases in ambulations were also noted during the first 5-minutes of assessment for females at 40 mg/kg/day onwards and during the 5 to 10-minutes of assessment for males at 80 mg/kg/day, compared with controls (P < 0.05). Additionally, statistically significant increases in rearing were observed during the first 10-minutes and 16 to 20-minutes of assessment for males at the high dose and during the first 5-minutes of assessment for females mid dose onwards, compared with controls (P < 0.05). This resulted in a statistically significant increase in overall rearing for males at the high dose, compared with controls (P<0.05). A statistically significant increase in total distance travelled was also observed during the first 5 or 10-minutes of assessment (females and males, respectively) for the high dose animals, compared with controls (P < 0.05), although overall distance travelled was not statistically significantly different from the controls. While these increases may indicate increased exploratory behavior, most individual values during at these time points were within the historical control ranges, and differences from controls were less evident as animals habituated to their surroundings. Although a possible relationship of these initial differences to the test article cannot be completely excluded, significant differences from controls were occasionally not dose-related or consistent in both sexes, overall changes in most parameters were not statistically significantly different from controls, and a number of individual animals from the test article-treated groups had values within the concurrent control ranges. Additionally, no associated signs of neurotoxicity nor any test histopathology correlations were observed for these animals. Considering these evidences, these changes were therefore not considered to represent an adverse effect of the test article.

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

Remarks on result:

other: no systemic adverse effect was observed

Critical effects observed:

no

Conclusions:

Under the study conditions, 42 days for males and 54 days for females NOAEL for repeated oral study is determined to be 80 mg/kg bw/day for systemic toxicity.

Executive summary:

A study was conduct to determine the repeated dose oral toxicity of the test substance, Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18-unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts) in rats according to OECD Guideline 422, in compliance with GLP. Four groups of 10 Crl:WI(Han) rats/sex/group were administered 0, 20, 40, or 80 mg/kg/day by daily oral gavage for 42 days for males (2 weeks prior to pairing, 1 week during the pairing phase, and 3 weeks post-pairing until the day before necropsy) and up to 54 days for females (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13) at a volume of 10 mL/kg. The following parameters were evaluated at regular intervals: mortality, clinical observations, bodyweights, food consumption, functional and behavioral assessments, estrous cycles, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Anogenital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. One pup/sex/litter/dose group was selected for recording of thyroid weights and processing of thyroids for microscopic examination.

Treatment at 80 mg/kg/day for males and 40 mg/kg/day for females resulted in occasional incidences of mouth rubbing, which generally occurred after. An isolated incidence of head burrowing was also noted for one male administered  80 mg/kg/day. These observations were considered to reflect the palatability or taste of the formulation and not an indication of the systemic toxicity. Administration
of 80 mg/kg/day was associated with a minor decrease in body weight gain for males at the start of dosing; no effect on food consumption was noted for these animals, and this finding was considered not adverse. Body weight and food consumption was unaffected for the remaining treated animals. Upon detailed weekly clinical assessment, occasional incidences of dose-related mild to moderate decreased activity were noted for males administered ≥40 mg/kg/day. No other signs of neurotoxicity were observed in these animals, and this finding was considered not representative of an adverse effect of treatment. Motor activity evaluations identified statistically significant increases in basic and fine
movements, ambulations, and/or rears for animals administered ≥40 mg/kg/day, compared with controls. A statistically significant increase in total distance travelled was also observed for animals administered 80 mg/kg/day, compared with controls. These changes were generally observed during the first 5 or 10-
minutes of assessment (females and males, respectively), and although they may be related to the test substance, they were considered not adverse. The test substance treatment did not change number and length of estrus cycles, mating, fertility, and fecundity. All pregnant females littered and maintained a live litter to LD 13. No effect of administrationwas evident on gestation length, litter size, or pup survival indices. No effects on anogenital distance, nipple counts, or thyroid hormones were observed in litters from the treated F0 females at any dose level, compared with controls. No-related clinical observations or macroscopic abnormalities were noted for offspring from treated groups. Differences in some clinical chemistry parameters in the treated F0 animals were minor, not dose related, specific to one sex only, had no microscopic correlations, and therefore were considered of no toxicological significance. No treatment-related macroscopic
or microscopic findings were recorded for F0 animals or pups and no changes in organ weight were apparent. Under the study conditions, the NOAEL for systemic toxicity was determined to be 80 mg/kg/day by the study author (Haroon, 2021).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

EC Number:

947-766-0

Molecular formula:

Since the test substance is a complex UVCB, no defined molecular formula is available.

IUPAC Name:

Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

Test material form:

solid: bulk

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty-three male and 46 female Crl:WI(Han) rats were obtained from Charles River Laboratories
Margate United Kingdom, in order to provide sufficient animals for study selection.
Upon arrival, males were 9 to 10 weeks old and females were 7 to 8 weeks old. At the start of dosing, males weighed between 274.8 and 380.7 g and were 11 to 12 weeks old, females weighed between 170.9 and 223.9 g and were 10 to 11 weeks old.
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for 2 weeks prior to initiation of dosing (males) or 1 week prior to smearing (females), and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.
Animals were housed in cages that conform to the Code of Practice for the Housing and Care of
Animals Bred, Supplied, or Used for Scientific Purposes (Home Office,#2014).
Animals were housed in groups (up to four animals/cage by sex [both sexes pre-pairing and males post-pairing] or with one female and one male [pairing]), individually (mated females), or with their litter (females during lactation).
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed by Covance for specific contaminants.
Animals had ad libitum access to SDS Rat and Mouse breeder diet VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.
Animals were provided with wooden Aspen chew blocks and rodent retreats. During gestation and
lactation, nesting materials (i.e. paper wool) were provided as forms of environmental enrichment.
Upon arrival, animals were assigned to dose groups using an internal randomization procedure. Animals were individually identified by electronic implant. Animals selected for FOB assessments were identified by tail markings.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was administered once daily orally by gavage. Males were dosed for 42 consecutive days (2 weeks prior to pairing, 1 week during the pairing phase, and 3weeks post-pairing until the day before necropsy). Females were dosed for up to 54 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD13).
A dose volume of 10 mL/kg was used. Individual dose volumes were based on the most recent individual body weights.

Details on mating procedure:

Animals were paired on the day following 15 days of dosing. During the pairing phase, one male was housed for up to 7 days with one female from the same dose group. Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon confirmation of mating, vaginal washing was discontinued, and the male was removed. The day on which mating was confirmed was designated as GD 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared for use in Week 1 and 6 were analyzed to determine the achieved concentration. Triplicate samples were taken from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of the control formulations and was analyzed.

Duration of treatment / exposure:

42 days for males and 54 days for females

Frequency of treatment:

Once daily

Details on study schedule:

Males were dosed for 42 consecutive days (2 weeks prior to pairing, 1 week during the pairing phase, and 3weeks post-pairing until the day before necropsy). Females were dosed for up to 54 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD13).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats/sex/group

Control animals:

yes, concurrent no treatment

Positive control:

No

Examinations

Parental animals: Observations and examinations:

HEALTH MONITORING
All animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.
CLINICAL EXAMINATIONS
Each animal was given a detailed physical examination daily (at a similar time on each day) from the first day of dosing until the day of necropsy.
POSTDOSE OBSERVATIONS
Initially, animals were observed daily prior to dosing and immediately after dosing upon to return to home cage until Pre-Pairing Day 14, inclusive. A few incidences of mouth rubbing following dosing upon return to the home cage following dosing were noted for animals administered 80 mg/kg/day and postdose opbservations were recommenced from Pairing Day 3 for males and Pairing Day 3 or GD0/1/2 for females.
BODY WEIGHTS
Male body weights were recorded once on the day prior to dose initiation, on the first day of dosing and at weekly intervals thereafter, and on the day prior to necropsy (Postpairing Day 21). A fasted terminal body weight was recorded at necropsy on Postpairing Day 22.
Female body weights were recorded once on the day prior to dose initiation; on the first day of dosing and weekly prior to pairing; on GD 0, 7, 14, and 20; and on LD 0 (where applicable) 1, 4, 7, and 13. A fasted terminal body weight was recorded at necropsy on LD 14.
FOOD CONSUMPTION
Male food consumption was determined once weekly prior to pairing and post-pairing until the day prior to necropsy.
Female food consumption was determined once weekly prior to pairing; from GD0 to 7, 7 to 14, and 14 to 20; and from LD 1 to 4, 4 to 7, and 7 to 13.
PARTURITION
Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25.

Oestrous cyclicity (parental animals):

Daily vaginal washings (lavage) were taken from all females for 14 consecutive days prior to dosing,
from the start of dosing until the confirmation of mating, and on the morning of LD 14, prior to necropsy.

Sperm parameters (parental animals):

Sections of testes and epididymides were stained with Periodic Acid-Schiff (PAS) for qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure.

Litter observations:

Litter size and sex:
On PND 1, 4, 7, and 13, litter size and pup sex were recorded. Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition were given a macroscopic necropsy.
On PND 4, litters were culled to 10 pups/litter, with five pups/sex, when possible. Culled pups had sex confirmation at necropsy and were discarded following completion of blood sampling.

Clinical observations:
Each pup was given a detailed clinical examination daily from PND 1.

Body weight:
On PND 1, 4, 7, and 13, pup body weights were recorded.

Anogenital distance:
On PND 4 postcull, the anogenital distance was measured.

Nipple/areolae count:
The number of nipples/areolae for male pups was counted on PND 13.

Functional observational battery:
All adult animals were assessed by detailed clinical observations, quantitative assessments, and elicited responses, including hindlimb foot splay and forelimb and hindlimb grip strength and motor activity determination.

Postmortem examinations (parental animals):

Blood samples for hematology, clinical chemistry and thyroid hormone assessments were collected at necropsy from all surviving adults. Organ weights were recorded and microscopic examinations were performed.

Postmortem examinations (offspring):

Complete necropsies were performed on all animals (except pups culled on PND 4), and any macroscopic abnormalities were noted. Additionally, samples were collected at necropsy from selected PND 4 and 13 pups for thyroid hormone assessments.

Statistics:

Analysis of variance (ANOVA) and pairwise comparisons, as applicable, were used to analyze the
following.
- Body weight (adult animals)
- Body weight change (adult animals)
- Food consumption
- Thyroid hormone analysis (male and female pups), where a single sample for each pup is obtained.
No analysis was performed for pooled samples.
Prior to the performing the ANOVA, Levene’s test was performed to test for equality of variances
between groups:
- Where Levene’s test was significant (P ≤ 0.05), a rank transformation (to stabilize the variances)
was applied before the ANOVA was conducted (note: Levene’s test was not applied to the rank-transformed data).
- Where Levene’s test was not significant (P > 0.05), ANOVA was conducted.

For comparisons to a single control:
- If the group effect of the ANOVA was significant (P ≤ 0.05), Dunnett’s test was used for pairwise
comparisons between each test article-treated and control group.
- If the ANOVA was not significant (P > 0.05), the Dunnett’s test results were not applicable to the
evaluation and were not reported or used to interpret study data.
Anogenital distance was analyzed using analysis of covariance (ANCOVA). Cube root litter weight
was taken as the covariate within the analysis.
Data containing values above/below the limit of quantitation were not analyzed, and the tables were footnoted accordingly. Where insufficient data were available for meaningful analysis, no analysis was performed, and the tables were footnoted accordingly.

Reproductive indices:

The following indices were determined: Mating Index (%), Fecundity Index (%) and Fertility Index (%)

Offspring viability indices:

The following indices were determined: Live Pups/Litter, liveborn, stillborn and uncertain.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Occasional incidences of mouth rubbing, which generally occurred after dosing were recorded for animals administered 80 mg/kg/day and females administered 40 mg/kg/day. An isolated incidence of head burrowing was also noted for one male administered 80 mg/kg/day. These observations were considered to reflect the palatability or taste of the formulation and not an indication of the systemic toxicity of the substance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Administration of 80 mg/kg/day was associated with a minor decrease in body weight gain for males at the start of dosing; no effect on food consumption was noted for these animals, and this finding was considered not adverse. Body weight and food consumption was unaffected for the remaining animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No effect on food consumption was noted for these animals, and this finding was considered not adverse. Body weight and food consumption was unaffected for the remaining treated animals.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared with controls, noteworthy or statistically significant changes in clinical chemistry parameters included minimally increased bile acids in females administered ≥20 mg/kg/day, minimally increased cholesterol in females administered 80mg/kg/day (P<0.05), minimally increased calcium in males administered ≥20 mg/kg/day (P<0.05), and minimally increased inorganic phosphorus (P<0.01) and minimally decreased chloride in females administered 80 mg/kg/day (P<0.05). These changes were minor, generally without dose-relationship, limited to one or the other sex, and, in the absence of any microscopic correlates, were considered of no toxicological significance.

Endocrine findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Compared with controls, statistically significantly increased basic and fine movements were observed during the first 5 or 10-minutes of assessment (females and males, respectively) at 80 mg/kg/day (P< 0.05). Statistically significant increases in ambulations were also noted during the first 5-minutes of assessment for females at 40 mg/kg/day onwards and during the 5 to 10-minutes of assessment for males at 80 mg/kg/day, compared with controls (P < 0.05). Additionally, statistically significant increases in rearing were observed during the first 10-minutes and 16 to 20-minutes of assessment for males at the high dose and during the first 5-minutes of assessment for females mid dose onwards, compared with controls (P < 0.05). This resulted in a statistically significant increase in overall rearing for males at the high dose, compared with controls (P<0.05). A statistically significant increase in total distance travelled was also observed during the first 5 or 10-minutes of assessment (females and males, respectively) for the high dose animals, compared with controls (P < 0.05), although overall distance travelled was not statistically significantly different from the controls. While these increases may indicate increased exploratory behavior, most individual values during at these time points were within the historical control ranges, and differences from controls were less evident as animals habituated to their surroundings. Although a possible relationship of these initial differences to the test article cannot be completely excluded, significant differences from controls were occasionally not
dose-related or consistent in both sexes, overall changes in most parameters were not statistically significantly different from controls, and a number of individual animals from the test article-treated groups had values within the concurrent control ranges. Additionally, no associated signs of neurotoxicity nor any test histopathology correlations were observed for these animals. Considering these evidences, these changes were therefore not considered to represent an adverse effect of the test substance.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: No adverse effects observed on fertility and reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

Remarks on result:

other: no adverse effects observed on viability, growth and development

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

A study was conduct to determine the repeated dose oral toxicity of the test substance, Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18-unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts) in rats according to OECD Guideline 422, in compliance with GLP. Four groups of 10 Crl:WI(Han) rats/sex/group were administered 0, 20, 40, or 80 mg/kg/day by daily oral gavage for 42 days for males (2 weeks prior to pairing, 1 week during the pairing phase, and 3 weeks post-pairing until the day before necropsy) and up to 54 days for females (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13) at a volume of 10 mL/kg. The following parameters were evaluated at regular intervals: mortality, clinical observations, bodyweights, food consumption, functional and behavioral assessments, estrous cycles, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Anogenital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. One pup/sex/litter/dose group was selected for recording of thyroid weights and processing of thyroids for microscopic examination.

Treatment at 80 mg/kg/day for males and 40 mg/kg/day for females resulted in occasional incidences of mouth rubbing, which generally occurred after. An isolated incidence of head burrowing was also noted for one male administered  80 mg/kg/day. These observations were considered to reflect the palatability or taste of the formulation and not an indication of the systemic toxicity. Administration
of 80 mg/kg/day was associated with a minor decrease in body weight gain for males at the start of dosing; no effect on food consumption was noted for these animals, and this finding was considered not adverse. Body weight and food consumption was unaffected for the remaining treated animals. Upon detailed weekly clinical assessment, occasional incidences of dose-related mild to moderate decreased activity were noted for males administered ≥40 mg/kg/day. No other signs of neurotoxicity were observed in these animals, and this finding was considered not representative of an adverse effect of treatment. Motor activity evaluations identified statistically significant increases in basic and fine
movements, ambulations, and/or rears for animals administered ≥40 mg/kg/day, compared with controls. A statistically significant increase in total distance travelled was also observed for animals administered 80 mg/kg/day, compared with controls. These changes were generally observed during the first 5 or 10-
minutes of assessment (females and males, respectively), and although they may be related to the test substance, they were considered not adverse. The test substance treatment did not change number and length of estrus cycles, mating, fertility, and fecundity. All pregnant females littered and maintained a live litter to LD 13. No effect of administrationwas evident on gestation length, litter size, or pup survival indices. No effects on anogenital distance, nipple counts, or thyroid hormones were observed in litters from the treated F0 females at any dose level, compared with controls. No-related clinical observations or macroscopic abnormalities were noted for offspring from treated groups. Differences in some clinical chemistry parameters in the treated F0 animals were minor, not dose related, specific to one sex only, had no microscopic correlations, and therefore were considered of no toxicological significance. No treatment-related macroscopic
or microscopic findings were recorded for F0 animals or pups and no changes in organ weight were apparent. Under the study conditions, the NOAEL for reproduction-developmental toxicity was determined to be 80 mg/kg/day by the study author (Haroon, 2021).