Reaction mass of Amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2019 to August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide

EC Number:

907-578-1

Molecular formula:

C9H14O4.2Li, C25H48NO3Li, C27H52NO3Li

IUPAC Name:

Reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 78 - 84 days (males), 71 - 77 days (females)
- Weight at study initiation: Males: 336 - 454 g; Females: 202 - 277 g;
- Housing: House 2 or 3 per cage by sex in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 22 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 23 - 66
- Air changes (per hr): 10 minimum
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 27 March 2019 To: 25 April 2019 (males) and 15-21 May 2019 (females and pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Propylene glycol 10% in Milli-Q Water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared as required and transferred to the animal unit immediately or stored in a refrigerator set to maintain 4°C, protected from light and dispensed daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by Ultra Performance Liquid Chromatography (UPLC) using a validated analytical procedure.

Duplicate sets of top, middle and bottom samples (duplicate middle only for control) were collected for analysis; triplicate top, middle and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples. Samples volumes were collected as follows: Groups 1 and 2: 0.2 mL into a 5 mL volumetric flask and Groups 3 and 4: 0.1 mL into a 10 mL volumetric flask. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 20%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of 10% for each group.

Duration of treatment / exposure:

Males: Males were dosed once daily for 29 days, starting from 14 days prior to mating.
Females: Females were dosed once daily from 14 days prior to mating, then continuing through gestation and lactation periods until the day prior to termination (LD 13). In this way female animals were dosed from Study Day 1 up to Day 54 (dependent on mating).

Frequency of treatment:

Daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the findings from two previous preliminary range-finding studies conducted in male and female (non-pregnant) rats.
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were subjected to detailed clinical observations weekly from Week -1.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once during pretreatment and daily during the dosing period. Body weight for non-pregnant animals are not reported during lactation (not collected).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day.
- Time schedule for examinations: Food consumption was quantitively measured weekly for both sexes from Week -1 until pairing for mating, and on GD 0 to 7, 7 to 14 and 14 to 20 and LD 1 to 7 and 7 to 13 for the mated females, where possible.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

OTHER: Ophthalmic Examinations
- Time schedule for examinations: Animals were subject to ophthalmic examinations once during pre-treatment (all animals) and once during Week 4 (males only) or the week before termination (females only).

OTHER: Detailed functional observations
- Time schedule for examinations: Detailed functional observations were conducted for all animals once during pretreatment and weekly throughout the dosing period for all adult animals. From Week 6 onwards, only selected females continued to have detailed functional observations.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals on test day 30
- Maternal animals: All surviving animals on lactation day 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weights: Brain, epididymis, adrenal glands, pituitary glands, prostate, thyroid/parathyroid glands, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus.
- Microscopic evaluation: Bone marrow, brain, cervix, epididymis, oesophagus, eye, adrenal glands, harderian gland, mammary gland, parathyroid glands, pituitary glands, prostate, salivary glands, seminal vesicles, thyroid glands, gut-associated lymphoid tissue, heart, femorotibial joint, kidney, colon, cecum, rectum, liver, lung, lymph nodes, optic nerve, sciatic nerve, ovaries, pancreas, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, tongue, trachea, ureter, urinary bladder, uterus, vagina.

Statistics:

Levene’s test was used to assess the homogeneity of group variances.
Datasets with at least 3 groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animal 4003M at 300 mg/kg/day displayed cranial twitches prior to dosing on Day 22. The animal was still considered fit for dosing and there were no further occurrences.

In addition, at 300 mg/kg/day sporadic observations were indicative of potential toxicity, including: irregular respiration rate, hunched posture (also observed in one occasion at 75 mg/kg/day), cold to touch, piloerection and abnormal gait. Due to the short duration and infrequent occurrence these observations were considered to be not adverse.

Ploughing behaviour and salivation (including wet fur on lower jaw), whilst most prominent at 300 mg/kg/day (and in particular in females during gestation), were considered to likely be response to dosing and not adverse or evidence of toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight or body weight gains in male or female animals over the course of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no appreciable effect on the food consumption of male animals. In female animals prior to mating a difference was observed between the food consumption at 150 mg/kg/day animals over Day 8-15 compared with both the control (14.5 g/animal/day verses 19.3 g/animal/day), and compared with the pre-dose values (17.8 g/animal/day). As this effect was not observed at 300 mg/kg/day it is considered unlikely to be associated with the administration of the test item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were test item changes in the ophthalmic observations following administration of the test item.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item related changes to haematology parameters in this study. There were no test item related changes to coagulation parameters in this study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a dose-related change in the concentration of circulating sodium levels, and whilst statistically significant, were within a typical rage for rats and were considered not adverse.

Other parameters attained statistical significance but did not follow a dose-related pattern and were considered spurious and not related to the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in the functional observations and behaviour following the administration of the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related organ weight differences.

There were additional organ weight differences, which were considered not to be test item related: In males, there were higher thyroid weights at 150 and 300 mg/kg/day, and higher liver weights at
75 and 300 mg/kg/day. In females, there were higher brain weights at 150 and 300 mg/kg/day. However, there was no dose response for any of these weight changes. There were higher spleen weights at 300 mg/kg/day, for which there was no microscopic correlate. There were individual organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related gross findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related microscopic findings.

The only microscopic finding of note was in males and consisted of an increased incidence of splenic haemopoiesis at 300 mg/kg/day which was considered not to be test item-related since there were no correlating haematology findings.
Other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Stimulating Hormone (TSH) and Thyroxine (T4):
In male animals (F0) lower levels of TSH were observed at 300 mg/kg/day (2.03 ng/mL at 300 mg/kg/day compared with 3.13 ng/ml in the male control animals). There was also a higher incidence of animals with concentrations of TSH Female animals (F0) were found with a lower level of TSH, a modest reduction was noted at 300 mg/kg/day (1.35 ng/mL compared with 1.64 ng/mL in the female control animals). There were considered to be no changes to concentrations of T4 attributed to the test item.
These micro changes in circulating thyroid hormone levels are considered to be of doubtful toxicological significance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Not determinable

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of Lithium-Azelate-Thickener by once daily oral gavage was well tolerated in Sprague Dawley (CD) rats. There was no evidence of test item toxicity. The No Observed Effect Level (NOEL) was considered to be 150 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) was 300 mg/kg/day in this study.

Executive summary:

A study to assess the toxicity of the substance in the rat after repeated oral administration and to provide initial information on possible effects on reproduction and/or development and neurotoxicity was conducted according to OECD 422 guideline. Three test groups and one control group, each containing 10 males and 10 females were used in the study. Males were treated for 2 weeks prior to mating until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation. The dose levels for the study were based on the findings from two previous preliminary range-finding studies conducted in male and female (nonpregnant) rats and were selected to be 75, 150 and 300 mg/kg/day where the high dose level was expected to induce minimal tolerated toxicity in the form of slight body weight effects in the parent animals and the low dose levels would be a no-effect level in the parents. No overt toxicity was produced in the rats at the dose levels tested. The no observed effect level (NOEL) was considered to be 150 mg/kg/day and the no observed adverse effect level was 300 mg/kg/day.