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Key value for chemical safety assessment

Effects on fertility

Description of key information

A study to assess the toxicity of the substance in the rat after oral administration and to provide initial information on possible effects on reproduction and/or development and neurotoxicity was conducted according to OECD 422. The NOAEL was determined to be >300 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Necropsy uterine examination was omitted in error which impacted on the ability to assess potential post-implantation losses. Retrospective examination of fixed uterine tissue was made to give some qualitative estimation of post-implantation loss.
GLP compliance:
yes
Limit test:
no
Justification for study design:
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 and the number of animals used was considered the minimum to properly assess the potential effects.

The dose levels selected for use in this study were based on the findings from two previous preliminary range-finding experiments conducted in male and female (non-pregnant) rats. Initially, a dose level confirmation phase consisting of two groups of five animals dosed examined the acute oral tolerance of the test item at both 600 mg/kg/day and up to the limit dose of 1000 mg/kg/day. During the second phase, three test groups and one control group, each containing 10 males and 10 females (0, 100, 200 and 300 mg/kg/day), were used to more fully determine the toxicity of the test item.
The administration of lithium-azelate-amine thickener by repeat-dose, once daily oral gavage was not tolerated at 1000 or 600 mg/kg/day due to sustained low or absent food consumption and resultant adverse clinical signs. However, administration of lower doses up to 300 mg/kg/day was well tolerated for up to 16 days and was associated with lower body weight gain (both sexes) and transiently lower food consumption (females only). An increase in the adrenal weight and reduction in thymus (males only), uterus (females only) and pituitary (females only) weight was evident, however were considered not to be adverse.
Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day. Consequently, an appropriate dose range for this OECD 422 study was considered 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 were expected to be a no-effect level in the parents.
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)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
10% in Milli-Q Water
Details on 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.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 10% propylene glycol was known from previous studies to form a good solution of the test item at the required concentrations
- Concentration in vehicle: Up to 60 mg/mL
- Amount of vehicle (if gavage): 5 ml/Kg
- Lot/batch no. (if required): A0296
- Purity:
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 nights maximum
- Proof of pregnancy: vaginal lavage referred to as day 0 / day 1 of pregnancy
- After 14 days of unsuccessful pairing the female will be treated as if mating had occured
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individual solid bottom cages
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.
Details on study schedule:
One parental and one offspring generation.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
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
Parental animals: Observations and examinations:
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 weights 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 pretreatment (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.
Oestrous cyclicity (parental animals):
Vaginal lavages were taken early each morning and the stages of oestrous observed were recorded over a 14 day period prior to test item administration, then continuing through pre-mating test item administration and the mating period.
Sperm parameters (parental animals):
Testes of all Group 1 and Group 4 males, examined for staging of spermatogenesis (qualitative evaluation).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 or 5 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD). Nipple retention in males.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals (8 rats/sex/group) were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

Offspring at scheduled termination will be examined for gross lesions, with samples of any abnormal tissues taken and fixed. All pups will be examined by open dissection for internal sex confirmation. Thyroid and parathyroid will be collected from one pup of each sex per litter. The remaining carcasses will be discarded.
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.
Reproductive indices:
For each group -
Fertility index (male) = Number siring a litter / Number paired
Fertility index (female) = Number pregnant / Number paired
Gestation index = Number bearing live pups / Number pregnant
Offspring viability indices:
For each litter and group -
Live Birth Index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0
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, fur erected 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.
Dermal irritation (if dermal study):
not examined
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 animals over the course of the study, nor in female animals prior to mating and over the gestation period.
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 sodium, 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
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 minro changes in circulating thyroid hormone levels are considered to be of doubtful toxicological significance.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no test item related changes in the duration or pattern of the oestrus cycle.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no test item effects on gestation length. Group means ranged from 21.8 to 22.1 days (typical for this strain of rat).

All pregnant females produced a live litter, giving a gestation index of 100% for all groups and with a similar ratio of male:female pups.

A difference in the total number of pups born was observed at 300 mg/kg/day (12.2 compared to 15.4 in the control). The lower mean value at 300 mg/kg/day was influenced by 2 animals with notably lower numbers of pups (3 pups for Animal 4506 and 5 pups for Animal 4507). A posthumous examination of the uterus (performed after histology sectioning) identified that there had been at least 9 and 10 implantation sites, respectively. Given the markedly higher number of implantation sites it was inferred that the lower number of pups in the litter was a result of post-implantation loss and not due to a reduction in the number of corpora lutea and implantation rates.

As part of the posthumous examination of the uteri, the rat at 150 mg/kg/day (3506) with 6 pups was found with 7 implantation sites. This indicates there was at least one post implantation loss. Overall however, there was considered to be no apparent reduction in the number of pups at 150 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Historically, the mean number of live pups on Lactation Day (LD) 0 ranges from 10.9 to 15.8 (average of 12.9). At 300 mg/kg/day the mean number of live pups on LD 0 (11.6) was within the lower end of the expected range for the Sprague Dawley Rat.

Survival rates for the pups were also typical for this strain of rat and there was no test item effect on the viability index of the pups over Lactation Day 0-4 or, following litter standardisation, between Lactation Days 4 and 13.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter-mean pup weight was similar over all groups in male pups and female pups. A smaller mean litter weight was observed at 300 mg/kg, however this was attributed to the slightly smaller litter size (see above) and not due to administration of the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no test item differences to the anogenital distance of male or female pups on PND 4
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item related effects on organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item related macroscopic findings.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item related microscopic findings.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Stimulating Hormone (TSH) and Thyroxine (T4):
In the pups (F1), there were no changes in T4 on PND 4 pooled samples or PND 13 samples (pooled or individual). Due to the high frequency of TSH samples
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Results of preliminary range-finding study:

In the dose level confirmation phase, dose levels of 1000 or 600 mg/kg/day produced reduced food consumption, leading to bodyweight losses and clinical signs of poor condition. Dosing was terminated however there were no improvements in the condition of the animals, and they were killed prematurely. Necropsy examination revealed findings of white/thick/gelatinous material in the stomach believed to be the test item formulation. There was also evidence of discolouration in the gastrointestinal tract.

Dose levels were lowered in Phase 2. In this phase there was a reduction in bodyweight gains in males at 200 and 300 mg/kg/day and females at 300 mg/kg/day. Food consumption was also lower in females at 300 mg/kg/day at the start of the dosing period. Organ weight differences were noted. Increased adrenal weights in males at 300 mg/kg/day, decreased uterus weights in females at 300 mg/kg/day, decreased pituitary weights in females at 300 mg/kg/day and decreased thymus weight in males and females at 300 mg/kg/day.

Based on the results from this study, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.

Conclusions:
In conclusion, administration of Lithium-Azelate-Thickener by once daily oral gavage was well tolerated in Sprague Dawley (CD) rats. There were no evidence of test item changes to mating and fertility, maternal behaviour or pre-weaning litter viability. 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 in this study.
Executive summary:

A study to assess the toxicity of the substance in the rat after 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 (non-pregnant) 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. There was no evidence of any test item related changes to mating and fertility, maternal behaviour or pre-weaning litter viability. 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A regulatory study conducted in accordance with OECD422 guideline and to GLP.
Additional information

A study to assess the toxicity of the substance in the rat after oral administration and to provide initial information on possible effects on reproduction and/or development and neurotoxicity was conducted according to OECD 422 guideline (Bain 2019).

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 (non-pregnant) 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. There was no evidence of any test item related changes to mating and fertility, maternal behaviour or pre-weaning litter viability. 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.

Justification for classification or non-classification

No effects on fertility or reproduction were encountered in rats treated orally at doses levels up to 300 mg/kg/day. This dose level was considered from previous range-finding studies to be the subtoxic dose which would be likely to induce tolerated toxicity in the form of slight body weight effects in rats. In the absence of effects on fertility or reproduction at the dose level of 300 mg/kg/day, the test substance was not classified as a reproductive toxicant.

Additional information