Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-14 to 2018-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

EC Number:

279-510-2

EC Name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Cas Number:

80584-99-2

Molecular formula:

n.a.

IUPAC Name:

Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Test material form:

liquid

Details on test material:

- Name: VOELOFA Monomer
- Batch no.: 3613676
- CAS no.: 80584-99-2
- Appearance: brown liquid
- Purity: 100%; LC/ELSD
- Expiry date: 2018-03-26
- Storage conditions: Fridge (2 - 8 °C); Keep away from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Calco (LE), Italy
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 175 to 200 g for males and 151 to 175 g for females
- Housing: From arrival to pairing, animals will be housed up to 5 of one sex to a cage. During mating, animals will be housed one male to one female in clear polysulfone cages. After mating, the males will be re-caged as they were before mating, the females will be transferred to individual solid bottomed cages.
- Diet (e.g. ad libitum): Yes, commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy)
- Water (e.g. ad libitum): Yes
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Vehicle:

olive oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC Study no. A2826). Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration.

Duration of treatment / exposure:

Males: 35/36 days
Females: 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice.

Frequency of treatment:

once a day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Medium

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

10 male and 10 female rats per dose. 4 dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a preliminary non-GLP compliant study (RTC no. E0178) where slight toxicity was observed in males at 1000 mg/kg/day.
- Rationale for animal assignment (if not random): The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Positive control:

n.a.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure were followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.
Animals judged to be in extremis were killed. A complete necropsy were performed in all cases.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters, where possible), 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters, where possible), 5 males and 5 females were randomly selected from each group and the motor activity were measured (for approximately 5 minutes) by an automated activity recording device

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly (whenever possible) from allocation to termination. Females were weighed weekly (whenever possible) from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams and pups were weighed on Days 1, 4, 7 and 13 post partum. Dams were also weighed just prior to necropsy.

FOOD CONSUMPTION : Yes
- Time schedule for examinations: weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked in table 2 were examined.

Sacrifice and pathology:

Parental animals and those that have completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia. Animals sacrificed for humane reasons were killed with carbon dioxide. Pups killed for humane reasons or those that have completed the scheduled test period (Day 14 post partum) were euthanised by intraperitoneal injection of Thiopenthal.

GROSS PATHOLOGY: Yes
The clinical history of the males and females of the parental generation were studied and a detailed post mortem examination were conducted (including examination of the external surface and orifices).

HISTOPATHOLOGY: Yes
The tissues required for histopathological examination are listed in table 3. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) were performed.

Organ Weights:
Parental animals
From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Pups at Day 14 post partum
Thyroid were weighed from one male and one female from each litter and preserved for possible histopathological examination (if required, additional cost). The thyroid weight were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups wereassessed by the non-parametric version of the Williams test. The criterion for statistical significance was p< 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation after dosing was recorded in two males at 300 mg/kg/day and in the majority of males receiving 800 mg/kg/day during the course of the study. One male in the high dose group was noted to have damaged ear.

Mortality:

mortality observed, treatment-related

Description (incidence):

Six cases of premature death occurred during the study.
Males: A single male animal from the control group was found dead on Day 20 of the mating phase.
Females: Five high dose females were humanely sacrificed on Day 0 post partum for difficulty in delivery.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant alterations were observed in body weight of males during the whole duration of treatment. However, at 800 mg/kg/day body weight gain was statistically significantly reduced on Day 8 of treatment. Significantly reduced body weight and body weight gain were observed at 800 mg/kg/day during gestation phase. Reductions in body weight and body weight gain were still evident in the high dose group during post partum even if only two dams were present in this phase.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced food consumption was recorded in the high dose group during the 2 weeks of treatment before starting the pairing phase (Days 8 and 15). At 100 and 300 mg/kg/day, a statistically significant decrease also occurred on Day 15 although the male control group showed a very high mean value of food consumption. At 300 and 800 mg/kg, food consumption of female animals was statistically and dose-dependently reduced during the 2 weeks of treatment before starting the pairing phase and on gestation Days 7 and 14.
During lactation period, a dose-related trend of reduced food consumption was noted when compared to controls. At 800 mg/kg/day the reduction was still evident even if only two dams were present in this phase.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Statistically significant decrease of erythrocytes, haemoglobin and haematocrit was recorded in male animals dosed at 800 mg/kg/day (approximately 7% below controls). Reticulocytosis was also recorded in the same group animals (57% in males and 22% in females). In addition, mean corpuscular haemoglobin concentration was decreased in females receiving 800 mg/kg/day (4% below controls). Due to the slight severity, the above findings were considered to be not adverse.
Coagulation: No effects were observed in the mean of prothrombin time in both genders.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phosphatase was increased in three males dosed at 800 mg/kg/day. Compared with mean control data, the increments were 4.3 to 6.8 fold. In addition, triglycerides were decreased in animals of both sexes of the same group. Compared with mean controls, changes were 39% in males and 77% in females. Increased urea was recorded in one female receiving 800 mg/kg/day (91% above controls). The other statistically significant changes recorded (albumin and phosphorus in males, chloride, calcium bile acids and sodium in females) were of minimal severity and/or not dose related, therefore they were considered of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The evaluation of neurobehavioural parameters after removal from the home cage and in the open arena did not indicate significant differences between control and test item treated groups in both genders, throughout the whole observation period. The statistical significance which occurred of the measurement of landing foot splay in males in the mid dose group (300 mg/kg/day) did not follow a dose-dependent pattern and therefore considered an incidental finding.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slight decrease in terminal body weight of high dose treated males was observed when compared to the controls. An increase, statistically significant or not, was recorded in the relative mean liver weight in high dose male animals (approximately 19% of relative mean liver weight) and in absolute and relative mean adrenals weight in mid- and high dose treated female animals (approximately 24% and 42% of relative mean adrenals weight, for mid- and high dose females, respectively).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Enlarged adrenals in three mid and three high dose females or small thymus in one low, four mid and one high dose females were observed in animals that completed the treatment period. The remaining sporadic changes such as single or multiple dark and/or red area/s depressed in the glandular region of the stomach of control and treated females or distended uterus with clear fluid in one high dose female were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Atrophy of thymus and hypertrophy of adrenal cortex were observed in a single instance in high dose females, when compared to the controls. Considering the reduced number of high dose females sacrificed at term and subjected to histopathological evaluation (two surviving females) and the findings observed in the unscheduled high dose females, a possible treatment-related effect on the above mentioned organs could not be excluded. The remaining sporadic lesions, reported in control and treated animals, were considered to be an expression of spontaneous/or incidental pathology or physiological changes, seen in this species and age in this kind of studies in our experimental conditions.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormones determination: Triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) were decreased in almost all males dosed at 800 mg/kg/day. Compared with controls, changes were 52% for T3, 73% for T4 and 48% for TSH. Four males receiving 300 mg/kg/day also showed decrease of both TSH and T4. Changes were approximately 70%. No relevant changes were recorded in circulating hormones of T3, T4 and TSH in male pups on Day 14 of age.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422, adverse effects were found after oral administration of the test item in male and female Sprague Dawley rats in the high dose group. In this group, treatment-related histopathological changes and mortality were noted. Moreover, effects were seen in the adrenals weight in mid and high dose treated female animals. Based on the results, the LOAEL is considered to be 300 mg/kg bw/day for both sexes.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), the test item, VOELOFA Monomer was administered orally to 10 male and 10 female Sprague Dawley rats/dose in olive oil by gavage at dose levels of 0, 100, 300 and 800 mg/kg bw/day at a constant volume of 5 mL/kg bw. The animals were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 35/36 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum.

Test item related mortality was observed in male and female rats. Salivation after dosing was recorded in two males at 300 mg/kg/day and in the majority of males receiving 800 mg/kg bw/day during the course of the study. The high dose females sacrificed for difficulty in delivery showed signs of discomfort like pale appearance, piloerection and cyphosis. Prolapse of vagina was also noted. No significant differences between the test and control group were noted in neurobehavioural parameters (grip strength, motor activity and reactivity to stimuli). Significant body weight changes were observed at 800 mg/kg bw/day during gestation phase. Reductions in body weight and body weight gain were still evident in the high dose group during post partumeven if only two dams were present in this phase. At 100 and 300 mg/kg bw/day, a statistically significant decrease in food consumption was observed in males and at 300 and 800 mg/kg bw/day for females.

During lactation period, a dose-related trend of reduced food consumption was noted when compared to controls, which was evident at 800 mg/kg/day. No toxicologically adverse effects were seen in haematology and clinical chemistry parameters. A slight decrease in terminal body weight of high dose treated males was observed when compared to the controls. An increase, statistically significant or not, was recorded in the relative mean liver weight in high dose male animals (approximately 19% of relative mean liver weight) and in absolute and relative mean adrenals weight in mid- and high dose treated female animals. Atrophy of thymus and hypertrophy of adrenal cortex were observed in a single instance in high dose females, when compared to the controls.

No treatment related changes were observed in oestrous cycle, reproductive parameters, pairing combination and mating performance. Clinical signs in pups noted in the control, low and mid-dose groups included cold to touch, small size and pale appearance. The incidence of these signs did not indicate any test item related-effect. At 800 mg/kg bw/day, only two dams survived until Day 14 post partum. No other treatment related effects were observed in the pups. Based on the results of this study, the LOAEL for repeated dose toxicity is considered to be 300 mg/kg bw/day. This study is classified as acceptable and satisfies the guideline requirement for an oral repeated dose toxicity study in rats.