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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A weight of evidence approach is adopted to address the potential toxicity of the substance to fertility and development. The available information from the experimental or predicted data on the substance and experimental study on the strcutural analogue indicate the following:

1. Systemic availability: Based on the BCF value estimation of individual constituents of test substance using BCFBAF v3.02 of EPIWEB v 4.11, the overall estimated BCF value for the test substance was considered to be 70.8 L/kg ww. Physico-chemical properties of the substance suggest overall low absorption potential through oral, dermal and inhalation routes. Therefore, based on overall evidence, the substance is assumed to have a low systemic absorption and low bioaccumulation potential.

2. Toxicological effects in repeated dose toxicity: The sub-chronic toxicity study with the structural analogue showed no adverse effects on the reproductive organs of either sex and overall low systemic toxicity.

3. Toxicological effect on reproduction-development: The combined repeated dose toxicity and reproduction-developmental toxicity screening study with the substance itself showed no adverse effect on the fertility and reproductive performance of the parenteral animals and no adverse developmental effect on the offsprings. Moreover, the pre-natal developmental toxicity study with the structural analogue did not show any adverse effect on the development of the offsprings at any tested dose. 

4. Endocrine disruption effect: The combined repeated dose toxicity and reproduction-developmental toxicity screening study with the substance did not show any evidence of endocrine-mediated adverse effect or adverse effect on the endocrine organs.

Therefore, considering the above weight of evidence, no further testing would be required for fertility and development, according to the specific rules for adaptation from Column 1 standard requirements under the REACH Annex IX.

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same 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 (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.

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).

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

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

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.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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

Critical effects observed:

no

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

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

Critical effects observed:

no

Reproductive effects observed:

no

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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From June 10, 2013 to July 18, 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:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Sex: females, nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: 206-258g.
- Fasting period before study: no
- Housing:
Acclimatization: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with stock males on a one-to-one-basis or two-to-one-basis in Macrolon cages.
Post-mating: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- 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: 10 June - 18 July 2013

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Method of formulation: Formulations (w/w) were prepared at least once every 8 days and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature when prepared 5 hour before dosing. When prepared more than 5 hours before dosing, in the refrigerator under nitrogen.
- 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). 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%.

Details on mating procedure:

- M/F ratio per cage: 1/1 or 2/1 (one or two females were cohabitated with one stockmale).
- Age at mating of the animals in the study: Approximately 12 weeks
- Length of cohabitation: until sufficient mated females had been obtained for each dose group.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Duration of treatment / exposure:

Duration of treatment: From Days 6 to 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Administration of test substance was 14 days.

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:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose levels were selected based on results of the dose range finding study (Project 502145)

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7).

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

FOOD CONSUMPTION
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy were recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 20, animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Organs examined: according to guidelines

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined
as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal
inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

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 Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 10) to determine
intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-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. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where: Viable fetuses affected / litter (%) = number of viable fetuses affected / litter x 100

Clinical signs:

no effects observed

Details on maternal toxic effects:

Details on maternal toxic effects:
MORTALITY: At 100 mg/kg, one female had to be killed in extremis on Day 8 post-coitum. This animal showed lethargy (severe), flat posture, piloerection and hypothermia. At necropsy, stomach abnormalities (reddish glandular mucosa, enlarged stomach, granular red-brown contents) were noted and 16 implantations in utero.

CLINICAL SIGNS: Treatment related clinical signs were noted at 100 mg/kg, and consisted of hunched posture (two females for 6-8 days), rales (two females for 3 days), piloerection (six females for 1-4 days), and lean appearance (two females for 6 days). In addition, the female that was euthanized in extremis showed lethargy (severe), flat posture, piloerection and hypothermia. Salivation seen after dosing for one female at 10 mg/kg and sixteen females at 100 mg/kg 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). Alopecia was noted for single animals of all groups. This finding 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. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

BODY WEIGHTS: At 100 mg/kg, body weights and body weight gain were decreased during treatment (statistically significant on most occasions). Additionally, for (gravid) uterus corrected weight gain was statistically significantly decreased. Almost all females at this dose showed a body weight loss on Day 9 post-coitum showing a clear recovery during the remainder of the treatment period for all but two females. These two females showed early resorptions only at necropsy. At 10 mg/kg, five females showed reduced body weight gains when compared to controls. This was considered due to their small litter sizes, and not considered treatment related.

FOOD CONSUMPTION: At 100 mg/kg, absolute and relative food consumption was lower during treatment (statistically significant on most occasions). The main effect was noted during Days 6-9 post-coitum and showed some recovery during the rest of treatment. For two females food consumption remained very low on Days 9-15 postcoitum. At 10 and 30 mg/kg, no toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

MACROSCOPIC EXAMINATION: At 100 mg/kg, there were two females that showed emaciation. In addition, the female that was euthanized in extremis showed stomach abnormalities (i.e. reddish glandular mucosa, enlarged stomach, granular red-brown contents).
The incidence of other incidental findings (alopecia and diaphragmatic hernia of the liver) was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend.

MATERNAL PREGNANCY DATA: There were 22, 21, 21, and 22 pregnant females in the control, 10, 30 and 100 mg/kg groups, respectively, with 22, 19, 21 and 19 litters available for evaluation. At 100 mg/kg, there were two females that had 100% post-implantation loss, consisting of early resorptions. This resulted in an high mean value for early resorptions (not statistically significant). This was considered a secondary test substance effect caused by the very low food consumption and body weight loss during post-coitum. At 10 mg/kg, the lower number of viable fetuses and implantation sites per litter were mainly due to lower number of corpora lutea (for 4/6 rats with lower number of viable fetuses). As this occurred before start of treatment, it was not considered a treatment related effect. In addition at this dose, two females showed a high rate of early resorptions and low rate of viable fetuses. These values remained within normal limits, and as this was not noted at 30 mg/kg, it was not considered toxicologically relevant.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL FINDINGS
LITTER SIZE
No treatment related effect on litter size was noted up to 100 mg/kg. The mean number of viable fetuses per litter was 12.3, 9.1, 11.9 and 11.7 in the control, 10, 30 and 100 mg/kg groups, respectively. The lower mean value for viable fetuses per litter at 10 mg/kg was mainly due to lower number of corpora lutea. As this occurred before start of treatment, it was not considered a treatment related effect.

SEX RATIO
There were no treatment-related effects on the sex ratio of the fetuses.

FETAL BODY WEIGHT
Fetal body weights were unaffected for treatment up to 100 mg/kg. Body weights of fetuses (sexes combined) were 3.5, 3.5, 3.5 and 3.4 grams for the control, 10, 30 and 100 mg/kg groups, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
EXTERNAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal external morphology. One external malformation was noted in this study. One fetus at 10 mg/kg had a small lower jaw and due to singly occurrence in the low dose group, this finding was not considered to be treatment related. No external malformations and developmental variations were observed in any of the other fetuses, thus it was considered that test substance treatment at dose levels up to 100 mg/kg had no effect on fetal external morphology.

VISCERAL MALFORMATIONS
There were no treatment related effects on fetal visceral morphology. Visceral malformations were observed in one fetus at 10 mg/kg and two fetuses from two litters at 100 mg/kg. The malformations that occurred in the high dose group were situs inversus and absent eye. Both these findings were seen previously in historical control fetuses and due to singly occurrence they were not considered to be treatment related. At 10 mg/kg, the affected fetus (the one with the small jaw externally) had a multiple cardiovascular abnormality characterized by malpositioned right subclavian artery (originating from the pulmonary trunk), narrow pulmonary trunk, absent ductus arteriosus and ventricular septum defect. This is an unusual abnormality (of the individual malformations, only the ventricular septum defect was seen in historical control fetuses), however, as it was noted in one fetus of the low dose group, it was not considered to be a test substance effect.

VISCERAL VARIATIONS
Visceral variations occurred in 9.8%, 6.9%, 13.1% and 13.1% of fetuses per litter in the control, 10, 30 and 100 mg/kg groups, respectively. Those observed in the test substance treated groups were small supernumerary liver lobe, liver appendix, pale spleen, dilated ureter, convoluted ureter, supernumerary artery, small thyroid and partially undescended thymus horn. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose relationship and/or occurred at frequencies within the historical control range.

SKELETAL MALFORMATIONS
There were no treatment related effects on fetal skeletal morphology. Skeletal malformations were observed in 3(3) fetuses (litters) of both the control and 100 mg/kg group. In the high dose group, two fetuses from two litters had a vertebral anomaly with or without associated rib anomaly. The one fetus with situs inversus viscerally, missed a lumbar centrum and arch and the finding in the other fetus was characterized by absence of a hemicentrum, arch and head of a rib. The latter fetus also had another skeletal malformation, namely severely malaligned sternebrae. The third skeletally malformed fetus in this group, had a rib anomaly (fusion of ribs). None of the above malformations were noted in concurrent controls, but two out of three (vertebral anomaly with or without associated rib anomaly, and severely malaligned sternebrae) were seen previously within the range of historical controls. Therefore, and due to the single occurrence of the rib anomaly, all skeletal malformations noted at 100 mg/kg were not considered to be treatment related.
Other skeletal malformations in this study only affected control fetuses. Bent limb bones in one fetus, malpositioned metatarsals in one fetus and costal cartilage anomaly in one fetus. Due to occurrence in the control group, these malformations were not related to treatment.

SKELETAL VARIATIONS
Skeletal variations occurred in 89.7% and 86.2% of fetuses per litter in the control and 100 mg/kg group, respectively. Of the variations at 100 mg/kg, statistically significantly decreased mean litter proportions were noted, when compared to concurrent control values, for reduced ossification of the skull (2.8% versus 7.5% per litter) and bent ribs (1.0% versus 9.9% per litter). However, for both groups, the incidences of reduced ossification of the skull and bent ribs were within their historical control ranges (2.7-21.2% per litter and 1.6-27.4% per litter, respectively). Consequently, these findings were considered not to be treatment related. Moreover, the decrease in the number of fetuses with reduced ossification of the skull and bent ribs at 100 mg/kg indicates that a greater proportion of fetuses in this group had complete ossification of the skull bones and ribs that were not bent, and this is not considered to be an adverse developmental effect.
Other variations noted in the high dose group were 14th rudimentary rib, 14th full rib, 7th cervical rudimentary rib, unossified sternebrae nos. 5 and/or 6, unossified sternebrae nos. 1,2,3 and/or 4, entire sternum unossified, slightly to moderately malaligned sternebrae, partially fused zygomatic arch, unossified hyoid, ossified vertebral centrum no. 1, reduced ossification of vertebral centra, reduced ossification of vertebral arches, unossified vertebral centra, unossified or reduced ossification of pubis, caudal shift of pelvic girdle and unossified metacarpals and/or metatarsals. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently and/or occurred at frequencies within the historical control range.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No abnormalities

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Conclusions:

Under study conditions, prenatal developmental toxicity maternal No Observed Adverse Effect Level (NOAEL) for test substance, solvent free sample was established to be 30 mg/kg bw/day. The developmental NOAEL is considered to be at least 100 mg/kg/ kg bw/day.

Executive summary:

A study was conducted to determine the developmental toxicity / teratogenicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride, according to a method similar to OECD Guideline 414, in compliance with GLP. Rats were exposed to the test substance once daily by oral gavage from Days 6 to 19 post-coitum at doses of 10, 30 and 100 mg/kg bw/day. Accuracy and homogeneity of formulations were demonstrated by analyses. On Day 8 post-coitum, one female had to be euthanized in extremis. This animal showed lethargy (severe), flat posture, piloerection and hypothermia. At necropsy, stomach abnormalities (reddish glandular mucosa, enlarged stomach, granular red-brown contents) were noted. Treatment-related clinical signs noted for eight surviving animals at 100 mg/kg bw/day consisted of hunched posture, piloerection and/or lean appearance. At 100 mg/kg bw/day, almost all females showed a body weight loss on Day 9 post-coitum with recovery during the remainder of the treatment period for all but two females; these two females showed early resorptions only at necropsy. For (gravid) uterus, corrected weight gain was also statistically significantly decreased. No substance-related toxicity was observed in the groups at 10 and 30 mg/kg bw/ day. No developmental toxicity was observed at any of the dose levels. Under the study conditions, the maternal systemic toxicity No Observed Adverse Effect Level (NOAEL) of the test substance was determined to be 30 mg/kg bw/day and the developmental toxicity NOAEL was considered to be 100 mg/kg/ kg bw/day (Wil research, 2013).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Fertility:

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).

Development:

A study was conducted to determine the developmental toxicity / teratogenicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride, according to a method similar to OECD Guideline 414, in compliance with GLP. Rats were exposed to the test substance once daily by oral gavage from Days 6 to 19 post-coitum at doses of 10, 30 and 100 mg/kg bw/day. Accuracy and homogeneity of formulations were demonstrated by analyses. On Day 8 post-coitum, one female had to be euthanized in extremis. This animal showed lethargy (severe), flat posture, piloerection and hypothermia. At necropsy, stomach abnormalities (reddish glandular mucosa, enlarged stomach, granular red-brown contents) were noted. Treatment-related clinical signs noted for eight surviving animals at 100 mg/kg bw/day consisted of hunched posture, piloerection and/or lean appearance. At 100 mg/kg bw/day, almost all females showed a body weight loss on Day 9 post-coitum with recovery during the remainder of the treatment period for all but two females; these two females showed early resorptions only at necropsy. For (gravid) uterus, corrected weight gain was also statistically significantly decreased. No substance-related toxicity was observed in the groups at 10 and 30 mg/kg bw/ day. No developmental toxicity was observed at any of the dose levels. Under the study conditions, the maternal systemic toxicity No Observed Adverse Effect Level (NOAEL) of the test substance was determined to be 30 mg/kg bw/day and the developmental toxicity NOAEL was considered to be 100 mg/kg/ kg bw/day (Wil research, 2013).

Justification for classification or non-classification

Based on the results of the combined repeated dose toxicity and reproduction-developmental toxicity screening study with the test substance and the pre-natal developmental toxicity study with the structural analogue, the substance does not warrant classification for fertility and development according to EU CLP regulation (EC) (1272/2008) criteria.