Cyclohexanaminium, N,N,N-trimethyl-, hydroxide (1:1)

Endpoint summary

Description of key information

NOAEL 150 mg/kg bw/d (highest dose tested due to corrosive properties of test substance)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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:

yes

Remarks:

Animal husbandry: Temperature was outside the target (maximum of 26.8 °C for approximately 30 minutes) on a single occasion during the study. The deviation did not noticeably affect the clinical condition of the animals or the outcome of the study.

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: yes (virgins)
- Age at study initiation: 12/13 weeks (males/females, respectively)
- Weight at study initiation: males 348-457 g, females 228-285 g
- Fasting period before study:
- Housing: One air-conditioned room in a barrier protected unit. Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages, in compliance with European Regulations (Directive 2010/63/EU).
- Diet (e.g. ad libitum): Rat pelleted complete diet ad libitum sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system or bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation period: 8 days between animal arrival and start of treatment (males) or start of pre-test oestrous cycle smears (females).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): >35
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a solution in the vehicle at concentrations of 7.5, 25 and 75 mg/mL, according to Standard Operating Procedures of the Test Facility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of preparations: two formulations were prepared at 5 and 100 mg/mL for stability purpose. Quadruple accurately measured 0.5 g samples were taken from each sampling position of each formulation according to the table below, immediately after preparation (Time 0) and after 6 hours (Time T+6) stored at ambient temperature. Other samples were immediately taken after preparation (Time 0) for the 8 days (T+8days) stored refrigerated (+2 to +8 °C) and the stored frozen (-15 to -25 °C) stability evaluation. The samples were then stored frozen (-15 to -25 °C) and analyzed.
Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours, in a refrigerator (2-8 °C) for at least 8 days, and in a freezer (≤ -15 °C) for at least 8 weeks.

Duration of treatment / exposure:

Males: 31 days, i.e. 14 days prior to mating, throughout the mating period and up to the day prior to necropsy. The first day of dosing is designated as Day 1.
Females that delivered were treated for at least 51 days, i.e. during 14 days prior to mating (the first day of dosing is designated as Day 1), the
variable time to conception (the first day of gestation is designated as G 0), the duration of the pregnancy and 13 days after delivery (the first day of birth is designated as L 0), up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for at least 41 days.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

= 15 mg a.i./kg bw/day

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

= 50 mg a.i./kg bw/day

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

= 150 mg a.i./kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Two dose-range finding studies were conducted.
In a 14-day pre-study oral gavage in the rat, 300 mg/kg active content (1500 mg/kg/day test compound) resulted in mortality of 1/3 female on Day 9 and in ulcerations of the stomach in all animals.
Therefore, half-lethal doses were tested in a dose range-finding study. Administration of Cyclohexanaminium, N,N,N, trimethyl , hydroxide administered in rats by oral gavage at dose levels of 250 and 750 mg/kg bw/day (corresponding to 50 and 150 mg/kg bw/d active substance) was well tolerated and did not induce any obvious toxicological effect.
The dose level of 750 mg/kg/day was therefore selected as the high dose level for the subsequent combined 28-day repeated dose toxicity study with reproduction and developmental toxicity screening (50 % of the lethal dose).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adults were observed twice daily, at the beginning and at the end of each working day (including weekends and public holidays). Offspring were examined once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed daily for clinical signs. To detect any clinical signs or reactions to treatment, the animals were observed before and at least once after dosing (based on information provided from the DRF phase). A full clinical examination was performed on each weighing day. This observation was made outside the home cage by placing the individual animals in a table. Towards the end of the gestation, females were examined daily for signs of parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Male and females were weighed at the time of randomization, on the first day of exposure (prior to the first exposure) and weekly thereafter during pre-mating and mating periods (for females, only pre-mating data are reported).
Mated females were weighed: on days 0, 4, 7, 11, 14, 17 and 20 of gestation and on days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION:
Food consumption of males was recorded weekly during the pre-mating period.
Food consumption of females was recorded for the following periods: weekly during the pre-mating period; on gestation days 0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20; and on lactation days 1 to 4, 4 to 7 and 7 to 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: First 5 animals/sex/group, at the end of the pre-mating period (Day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, for about 16 hours
- Parameters checked: Haemoglobin, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Packed cell volume, Red blood cell count, Mean corpuscular volume, Reticulocyte count, Platelet count, Total white blood cell count, Differential white blood cell count. Coagulation parameters examined: Prothrombin time, Activated partial thromboplastin time, Fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: First 5 animals/sex/group, at the end of the pre-mating period (Day 14).
In addition, in order to assess the effect of the test item on clinical chemistry parameters after 4 weeks of dosing, a new analysis of the
parameters listed below was performed for at least four animals per sex per group on the remaining serum samples initially taken for potential TSH or T4 hormones analysis, stored deep-frozen (between -70 and -90 °C) and analyzed within 4 months
- Animals fasted: Yes, for about 16 hours
- Parameters examined: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Total cholesterol, Triglycerides, Total bilirubin, Total protein, Albumin, Globulin, Albumin/globulin ratio, Creatinine, γ-Glutamyltranspeptidase, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase, Bile acids

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: on Day 31 (last day of dosing), the day before scheduled necropsy. Females: on PND 12 (penultimate day of dosing) or PND 13 (last day of dosing), 2 or 1 day(s) before scheduled necropsy, respectively.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity


OTHER:
- Estrous Cycles
- Mating, Pregnancy and Parturition Parameters
-Thyroid hormone analysis (total T4)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes


HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Compared with the concurrent controls, there was a dose-related increased incidence of animals showing hypersalivation associated or not with abnormal foraging and/or pedalling in 250 and 750 mg/kg bw/day groups for both sexes. This effect was considered to be a physiological response to the high pH value of the test compound rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. just after dosing).
In addition, noisy breathing was noted on gestation day 1 for 1 female each in the 750 mg/kg bw/day and 75 mg/kg bw/day groups. These isolated findings were considered incidental.
Other incidental findings included sores, scabs, and localized hairloss.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males, there was no test item-related effect on mean body weight and mean body weight gain for males. The mean body weight gain was comparable or slightly higher to that of control group in all treated groups.
In females, there was no adverse effect of the test item on mean body weight gain during the premating, gestation or lactation periods.
Mean body weight gains were slightly lower in all treated groups when compared with control on the first week of dosing and during gestation and lactation periods. However, as it occurred without dose relationship and had no impact on mean body weights at the end of the pre-mating period, gestation period or on terminal body weights in any treated groups, this was considered as not toxicologically relevant (mean terminal body weights ranged from 0.6 to 3.5 % lower than control group).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption was slightly higher to that of control in all treated groups for both sexes during the pre-mating period. However, the differences were minor and were therefore not considered to be of toxicological relevance.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences noted in haematological and coagulation parameters between control and treated rats of both sexes that were considered to be toxicologically relevant.
Although differences in mean values attained statistical significance compared with the concurrent controls, the higher in eosinophil count and percentage for females, the increase in white blood cells concentration and the decrease in haemoglobin concentration and packed cell volume for males were of no toxicological significance since it occurred without a dose-related trend and/or in view of its low magnitude.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

End of premating period: There were no differences noted in serum clinical chemistry parameters between control and treated rats of both sexes after two weeks of dosing that were considered to be toxicologically relevant.
Mean bile acid concentrations were higher in all treated females of all groups (25.06, 29.72 and 32.31 μmol/L in the 75, 250 and 750 mg/kg bw/day groups, respectively), compared with the concurrent control (18.12 μmol/L). The effect was exacerbated by one female in each the 250 and 750 mg/kg bw/day groups (nos. 55 and 74, respectively) with a bile acids concentration superior to 60 μmol/L. After exclusion of these 2 females, mean values in both groups (16.97 and 23.85 μmol/L in the 250 and 750 mg/kg bw/day groups, respectively) were comparable with the control group. This finding was therefore considered of no toxicological relevance.
Day of necropsy: Clinical chemistry parameters analysis performed on blood taken on the day of necropsy from adult males and females dosed for at least 4 weeks confirmed that there was no effect on any clinical chemistry parameters for both sexes that were considered to be toxicologically relevant. Although differences in mean values attained statistical significance compared with the concurrent control, the higher creatinine concentration for males or the lower albumin concentration for females were considered of no toxicological significance since there was no dose-related trend and/or in view of the low magnitude. In addition, mean values were within
the historical control range.
Thyroid hormone analysis: No differences in Total T4 levels were noted among the different groups of adult males or among the different groups of PND 13 pups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no test item-related effect on functional tests (auditory reflex, pupillary reflex, righting reflex and grip test) in any group for both sexes.
The variation in motor activity (motor monitor) did not indicate a relation with treatment.
The grip strength mean values were slightly lower than control for females treated at 75 and 250 mg/kg bw/day and higher than controls for treated males. This was considered to be due to intra-group variability and therefore incidental.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

When compared with controls, there were no organ weight differences that might indicate any relationship to treatment. Occasional organ weight changes including those with statistical significance observed in the testes and in adrenal glands and spleen for females were considered to be incidental or only to reflect normal individual variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no observations that were considered to be associated with administration of the test item at any dose level. All macroscopic findings were considered to be incidental and part of the normal background commonly observed in rodents.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

When compared with control, there was no evidence of treatment-related microscopic effects associated with oral administration (gavage) of the test item at 750 mg/kg/day. The nature or incidence of other histological findings in the organs examined did not indicate any relationship to treatment and they were considered to be incidental or related to some aspect of experimental manipulation other than administration of the test item.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at highest dose tested (half-lethal dose)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at highest dose tested (half-lethal dose)

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available and the criteria laid down in Regultion (EC) 1272/2008 (CLP), non-classification of the test substance regarding specific target organ toxicity is warranted.