Registration Dossier

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Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From March 26, 2013 to June 26, 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA study
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old).
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 153 grams; females: 122 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 26 March - 26 June 2013
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
From Day 1 to 30, formulations (w/w) were prepared daily within 5 hours prior to dosing. From Day 31 onwards, formulations (w/w) were prepared for a maximum of 8 days prior to dosing. Formulations were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

DOSE VOLUME:
10 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations in Weeks 1, 6 and 13). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and stability over 8 days in the refrigerator in the dark were also determined (highest and lowest concentration, in week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 28-day oral study with the test substance by daily gavage in the rat (information provided by the sponsor)
Positive control:
Not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: at pretest: all animals (including spare animals), at Week 13: Groups 1 and 4

ESTROUS CYCLE DETERMINATION
- Time schedule for examinations: Day 69 up to and including Day 90 of treatment.
- How many animals: All females, except for one female at 0 mg/kg (no vaginal lavage was possible, since the vagina was not open)

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines .

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: during Week 12/13 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
No mortality occurred that was considered to be related to treatment with the test substance. One animal at 100 mg/kg died at blood sampling at the end of treatment. This was considered not to be related to treatment.

CLINICAL SIGNS
Treatment at 100 mg/kg resulted in the following clinical signs:
- Rales in 5/10 males from Week 8 onwards and in 4/10 females from Week 4 onwards,
- Hunched posture in 1/10 males in Weeks 8 to 10, in 2/10 females in Weeks 7 to 10 and in all females from Week 13 onwards.
Lethargy, flat posture, shallow respiration and ptosis shown by one male at 100 mg/kg on a single day only in Week 4 of treatment was considered unrelated to the test substance, given the incidental occurrence and absence of similar findings among all other animals of this dose group.
Salivation seen after dosing among animals of the 100 mg/kg dose group from Week 3 of the treatment period onwards was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance and/or may be related to the microscopic observation of prominent intercalated ducts in the mandibular salivary glands.
No other treatment related clinical signs were observed. Incidental findings that were noted included scabs (neck), chromodacryorrhoea, rales (females at 30 mg/kg) and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
At 100 mg/kg, body weights and body weight gain were lower than controls in males and females achieving a level of statistical significance on most occasions. Body weight gain was lower from Week 2 onwards and in females from Week 4 onwards. Group mean body weight after 13 weeks of treatment was approximately 20 and 12% lower than control mean weight for males and females, respectively. At 10 and 30 mg/kg, body weights were similar to controls throughout treatment.

FOOD CONSUMPTION
At 100 mg/kg, a trend towards slightly lower food consumption was noted for males. When corrected for body weight, food intake values were similar to control levels. Food consumption of males and females at 100 mg/kg remained within normal levels for rats of this age and strain. At 10 and 30 mg/kg, food intake was similar to control levels.

OPHTHALMOSCOPIC EXAMINATION
There were no toxicologically relevant ophthalmology findings at pre-dose and in Week 13. Incidental ophthalmology findings consisted of (focal) corneal edema or opacity, pinpoint corneal opacities, and haemorrhage from hyaloid vessel or iris. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain.

ESTROUS CYCLE DETERMINATION
At 100 mg/kg, two females showed an acyclic estrous cycle of 11 or 14 days. All other females at 100 mg/kg, and all females of the control group and the 10 and 30 mg/kg groups showed a normal (regular) estrous cycle of 4 to 5 days during the period in which estrous cycle length was determined (Day 76 up to and including Day 90).

HAEMATOLOGY
The following statistically significant changes in haematology parameters distinguished treated animals from control animals:
- Lower relative reticulocyte counts in males at 30 and 100 mg/kg and females at 100 mg/kg,
- Lower haemoglobin and haematocrit level in females at 100 mg/kg.
The slightly higher relative neutrophils and lower relative lymphocyte counts in females at 100 mg/kg and the lower white blood cell counts (WBC) in both sexes at 10 mg/kg remained within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males and females at 100 mg/kg and in males at 30 mg/kg,
- Lower albumin and total protein level in males and females at 100 mg/kg,
- Higher urea level in males at 100 mg/kg,
- Higher glucose level in females at 100 mg/kg,
- Lower potassium level in males at 100 mg/kg,
- Lower chloride and calcium level in females at 100 mg/kg,
- Higher inorganic phosphatase level in males and females at 100 mg/kg (not statistically significant in females).
The higher alanine aminotransferase activities and lower albumin and total protein levels were generally outside the normal range for rats of this age and strain, while other changes remained within this range.
The higher sodium level in males at 10, 30 and 100 mg/kg was considered to be of no toxicological significance as these changes occurred in the absence of a dose-related trend, and remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR
Males and females at 100 mg/kg showed a reduced motor activity (both total movements and ambulations; only statistically significant for females).
One animal at 30 mg/kg showed an abnormal pupillary reflex in the right eye. As this was only one animal, this finding was considered to be of no toxicological relevance. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all other examined animals. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
The following (statistically significant) changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
- Slightly increased spleen weight and spleen to body weight ratio in males and females at 100 mg/kg (not statistically significant for absolute weights),
- Slightly lower seminal vesicles weights in males at 100 mg/kg.
Other statistically significant changes in organ weights at 100 mg/kg were attributed to the lower terminal body weight and occurred without correlating morphological findings. These changes consisted of higher brain to body weight ratio in males and females, lower heart weight and higher heart to body weight ratio in males and/or females, lower liver weight (not statistically significant in females) and higher liver to body weight ratio in males and females lower kidney weight in males and females, lower prostate weight, lower adrenal to body weight ratio in males, , higher epididymides and testes to body weight ratio, lower ovary weight and higher uterus to body weight ratio , lower thyroid and thymus weight in males .

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, anddid not show a clear dose-related incidence trend.


HISTOPATHOLOGY
Treatment-related microscopic findings were present in:
Trachea:
- Loss of cilia/loss of mucous cells was recorded at 100 mg/kg (Group 4) in 4/10 males (2: minimal, 1: moderate, 1: marked) and in 4/10 females (1: minimal, 1: slight, 2: moderate) and in females at 30 mg/kg (Group 3) in 3/10 animals (minimal).
- Hypertrophy/hyperplasia of the epithelium was recorded at 100 mg/kg in 2/10 males at a minimal degree and in 3/10 females (2: minimal, 1: slight) and in females at 30 mg/kg in 3/10 animals (2: minimal, 1: slight).
Lung (males):
- Alveolar macrophage foci (in some animals with a foamy appearance) were recorded at an increased incidence and/or severity at 100 mg/kg (Group 4) in 9/10 males (4: minimal, 5: slight) and at 30 mg/kg (Group 3) in 1/10 males (moderate). A background level of this finding was recorded in 1/10 males and 2/10 females of the control Group 1 and in 4/10 females at 100 mg/kg (Group 4), all at a minimal degree.
Duodenum:
- Hypertrophy of the duodenal villi was recorded at 100 mg in 2/10 males (1: minimal, 1: slight) and in 2/10 females (minimal).
Seminal vesicles:
- Vacuolar degeneration of the epithelium was recorded at 100 mg/kg (Group 4) in 10/10 males (5: minimal, 5: slight) and at 30 mg/kg (Group 3) in 2/10 males (minimal).
Thyroid gland (females):
- Minimal vacuolation of the follicular epithelium was recorded in 5/10 females at 100 mg/kg (Group 4).
Adrenal glands:
- Vacuolation of the zona glomerulosa was recorded at 100 mg/kg in 2/10 males (1: minimal, 1: slight) and in 7/10 females (4: minimal, 3: slight).
Mesenteric lymph node:
- Macrophage foci were recorded at an increased incidence and severity at 100 mg/kg (Group 4). This was recorded in 10/10 males (7: slight, 3: moderate) and in 10/10 females (1: minimal, 6: slight, 3: moderate). The incidences and severities of this finding recorded in the remaining dose Groups were considered to be within background: Control Group 1: 4/10 males and 4/10 females (minimal), 10 mg/kg (Group 2): 3/10 males (1: minimal, 2: slight) and 4/10 females (minimal) and at 30 mg/kg (Group 3): 6/10 males (5: minimal, 1: slight) and 5/10 females (4: minimal, 1: slight).
Salivary glands (mandibular):
- Prominent intercalated ducts were recorded at 100 mg/kg in 5/10 males (3: minimal, 2: slight) and in 6/10 females (4: minimal, 2: slight).
Spleen:
- Congestion was recorded at an increased incidence and severity at 100 mg/kg (Group 4) in 8/10 males (4: minimal, 3: slight, 1: moderate) and in 8/10 females (3: minimal, 4: slight, 1: moderate). A background level of this finding was recorded in 1/10 males of control Group 1 and in 1/10 males and 1/10 females at 30 mg/kg (Group 3) ), all at a minimal degree.
Bone marrow (sternum):
- Increased adipocytes in the sternal bone marrow at a slightly higher incidence and/or severity was recorded at 100 mg/kg (Group 4) in 5/10 males (minimal) and 4/10 females (2: minimal, 2: slight) and at 30 mg/kg (Group 3) in 5/10 females (3: minimal, 2: slight). A background level of this finding was recorded in 2/10 females of the control Group 1, 1/10 males and 3/10 females at 10 mg/kg (Group 2) and 1/10 males at 30 mg/kg (Group 3), all at a minimal degree.
Harderian glands (females):
-An increased amount of brown (porphyrin) pigment was recorded in females at 100 mg/kg (Group 4). This was seen in 7/10 animals (5: minimal, 2: slight). A background level of this finding was recorded in 2/10 males of the control Group 1, 3/10 females at 10 mg/kg (Group 2), 3/10 females at 30 mg/kg (Group 3) and 4/10 males at 100 mg/kg (Group 4), all at a minimal degree.

The findings recorded in trachea, lung (males), seminal vesicles and mesenteric lymph nodes are considered to be adverse microscopic findings at the incidences and severities recorded in this study. For the findings in duodenum, thyroid gland (females), adrenal glands and salivary glands adversity can’t be excluded.
The findings recorded in the spleen, sternal bone marrow and Harderian glands (females) can also be present as normal background findings in rats of this age and strain. Therefore, these findings, at these incidences/severities and in absence of any other indicators of toxicity in these organs, were not considered to be adverse.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.
Spermatogenic staging profiles were normal for all males examined.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
other: Non systemic effects driven by malnutrition due to test substance
Key result
Critical effects observed:
not specified

No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 in Weeks 1 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The concentrations analysed in the formulations of Group 2, 3 and 4 in Week 13 showed slightly higher values than the target concentrations (i.e. the mean accuracies for Group 2, 3 and 4 were 115, 110 and 117%, respectively). No explanation was found for this. The formulations of Group 2 and 4 in Weeks 1, 6 and 13 were homogeneous (i.e. coefficient of variation ≤10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours and were found to be stable in the refrigerator in the dark for at least 8 days. The long term storage samples were stable at ≤-70°C for at least 41 days.

Conclusions:
Under the study conditions, 90 d NOAEL for repeated oral study is determined to be 10 mg/kg bw/day.
Executive summary:

A 90-d study was conducted to determine the repeated dose oral toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3 -propanediammonium chloride in rats, according to OECD Guideline 408, in compliance with GLP. Dose levels were selected by the sponsor at 0, 10, 30 and 100 mg/kg bw/day, based on the results of a 28 -day study with the test substance. The test substance, formulated in water, was administered daily for at least 90 d by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. The following parameters were evaluated at regular intervals: clinical signs, functional observation parameters, body weight, food consumption, ophthalmoscopic parameters, clinical pathology, organ weights, macroscopic examination and histopathology of a selection of tissues. No treatment-related mortality occurred in the study. Treatment at 100 mg/kg bw/day resulted in hunched posture mainly at the end of treatment, slight decrease in motor activity, a slightly lower food consumption and body weight primarily for males. Histopathological findings at 100 mg/kg bw/day consisted of loss of cilia/loss of mucous cells in the trachea, hypertrophy/hyperplasia of the epithelium in the trachea, hypertrophy of the villi of the duodenum, vacuolation of the follicular epithelium of the thyroid gland (females only), vacuolation of the zona glomerulosa of the adrenal gland, vacuolar degeneration of the epithelium of the seminal vesicles, increased incidence and severity of macrophage foci in the lungs (males only) and mesenteric lymph node and prominent intercalated ducts in the mandibular salivary glands. Changes in blood parameters at 100 mg/kg bw/day consisted of lower relative reticulocyte counts in both sexes, lower haemoglobin and haematocrit level in females, higher alanine aminotransferase activity and inorganic phosphatase level and lower albumin and total protein level in both sexes, higher urea and lower potassium level in males, and higher glucose and lower chloride and calcium level in females. Histopathological findings at 30 mg/kg bw/day consisted of loss of cilia/loss of mucous and hypertrophy/hyperplasia of the epithelium in the trachea (females only), vacuolar degeneration of the epithelium of the seminal vesicles, increased severity of macrophage foci in the lungs (in one male only). Changes in blood parameters at 30 mg/kg bw/day were confined to lower relative reticulocyte counts and higher alanine aminotransferase activity in males. Other treatment-related microscopic findings consisted of increased incidence and severity of congestion of the spleen at 100 mg/kg bw/day (correlating with increased spleen weights), increased incidence/severity of adipocytes in the sternal bone marrow at 30 (females) and 100 mg/kg bw/day and increased incidence and severity of brown (porphyrin) pigment Harderian glands in females at 100 mg/kg bw/day. These findings can also be present as normal background findings in rats of this age and strain. Therefore, at these incidences/severities and in absence of any other indicators of toxicity in these organs, these findings were not considered to be adverse. Two out of ten females at 100 mg/kg bw/day showed an abnormal (acyclic) estrous cycle, which is above the incidence reported in historical control data. Histopathological examination of the female reproductive organs did not show treatment-related lesions. The observation of an acyclic period was seen in two animals of the high dose group showing apparent toxicity, and within this group the two affected animals also showed the greatest effect on body weight. An indirect effect following non-specific systemic toxicity or malnutrition is the most likely cause for the observed acyclic estrous period in these animals. The effects of lower seminal vesicle weight seen at 100 mg (only absolute, not relative, so linked to lower BW) and vacuolar degeneration of the epithelium of the seminal vesicles at 30 and 100 mg/kg bw/day are not considered to have reproductive effects considering that spermatogenic staging profiles were normal in all dose groups. Under the study conditions, the NOAEL for repeated dose toxicity is determined to be 10 mg/kg bw/day (Wil research, 2014).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Good quality

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose oral:


A 90-d study was conducted to determine the repeated dose oral toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3 -propanediammonium chloride in rats, according to OECD Guideline 408, in compliance with GLP. Dose levels were selected by the sponsor at 0, 10, 30 and 100 mg/kg bw/day, based on the results of a 28 -day study with the test substance. The test substance, formulated in water, was administered daily for at least 90 d by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. The following parameters were evaluated at regular intervals: clinical signs, functional observation parameters, body weight, food consumption, ophthalmoscopic parameters, clinical pathology, organ weights, macroscopic examination and histopathology of a selection of tissues. No treatment-related mortality occurred in the study. Treatment at 100 mg/kg bw/day resulted in hunched posture mainly at the end of treatment, slight decrease in motor activity, a slightly lower food consumption and body weight primarily for males. Histopathological findings at 100 mg/kg bw/day consisted of loss of cilia/loss of mucous cells in the trachea, hypertrophy/hyperplasia of the epithelium in the trachea, hypertrophy of the villi of the duodenum, vacuolation of the follicular epithelium of the thyroid gland (females only), vacuolation of the zona glomerulosa of the adrenal gland, vacuolar degeneration of the epithelium of the seminal vesicles, increased incidence and severity of macrophage foci in the lungs (males only) and mesenteric lymph node and prominent intercalated ducts in the mandibular salivary glands. Changes in blood parameters at 100 mg/kg bw/day consisted of lower relative reticulocyte counts in both sexes, lower haemoglobin and haematocrit level in females, higher alanine aminotransferase activity and inorganic phosphatase level and lower albumin and total protein level in both sexes, higher urea and lower potassium level in males, and higher glucose and lower chloride and calcium level in females. Histopathological findings at 30 mg/kg bw/day consisted of loss of cilia/loss of mucous and hypertrophy/hyperplasia of the epithelium in the trachea (females only), vacuolar degeneration of the epithelium of the seminal vesicles, increased severity of macrophage foci in the lungs (in one male only). Changes in blood parameters at 30 mg/kg bw/day were confined to lower relative reticulocyte counts and higher alanine aminotransferase activity in males. Other treatment-related microscopic findings consisted of increased incidence and severity of congestion of the spleen at 100 mg/kg bw/day (correlating with increased spleen weights), increased incidence/severity of adipocytes in the sternal bone marrow at 30 (females) and 100 mg/kg bw/day and increased incidence and severity of brown (porphyrin) pigment Harderian glands in females at 100 mg/kg bw/day. These findings can also be present as normal background findings in rats of this age and strain. Therefore, at these incidences/severities and in absence of any other indicators of toxicity in these organs, these findings were not considered to be adverse. Two out of ten females at 100 mg/kg bw/day showed an abnormal (acyclic) estrous cycle, which is above the incidence reported in historical control data. Histopathological examination of the female reproductive organs did not show treatment-related lesions. The observation of an acyclic period was seen in two animals of the high dose group showing apparent toxicity, and within this group the two affected animals also showed the greatest effect on body weight. An indirect effect following non-specific systemic toxicity or malnutrition is the most likely cause for the observed acyclic estrous period in these animals. The effects of lower seminal vesicle weight seen at 100 mg (only absolute, not relative, so linked to lower BW) and vacuolar degeneration of the epithelium of the seminal vesicles at 30 and 100 mg/kg bw/day are not considered to have reproductive effects considering that spermatogenic staging profiles were normal in all dose groups. Under the study conditions, the NOAEL for repeated dose toxicity is determined to be 10 mg/kg bw/day (Wil research, 2014).


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).

Justification for classification or non-classification

Based on the results of the 90 d repeated dose study with the read across substance (mainly displaying non-specific systemic toxicity), the substance does not warrant classification according to EU CLP (1272/2008/EC) criteria.