[No public or meaningful name is available]

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:

Experimental Phase: 05 January 2018 to 09 October 2018. Report Issue:

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Deviations:

yes

Remarks:

There were some minor deviations from the study plan. These deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Molecular formula:

not applicable as UVCB

IUPAC Name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males 70 to 77 days, females 84 to 91 days
- Weight at study initiation: Males 339 to 422g, females 246 to 301g
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid were used with the exception of 1) Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods 2) Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum (potable water from the public supply)
- Acclimation period: Males: seven days before commencement of treatment. Females: 21 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis were available for the diet and water. All diet and water used on the study was considered to be of acceptable quality and not to have interfered with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): of 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.
- Environmental Enrichment: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary. A plastic shelter was provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cage.

IN-LIFE DATES: From: To: 02 May 2018 (animal arrival) to 12 July 2018 (final necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

The test item was administered daily by gavage using a graduated syringe and rubber catheter inserted via the mouth once daily at approximately the same time of day. Females were not dosed if parturition was in progress at the scheduled time of administration. Animals were treated at constant doses in mg/kg/day. Dose volume was 5 mL/kg body weight calculated from the most recently recorded scheduled body weight.

The control group was treated in an identical maner with vehicle at the same volume as the treated group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix. Formulations were confirmed to be homogeneous and stable for the duration of use on the study.

The mean concentrations of the test substance in test formulations analysed during the study were within ±10% of the nominal concentration, confirming the accuracy of formulation. The difference from the mean value remained within 4%, confirming precise analysis.

Duration of treatment / exposure:

Approximately five weeks for males including two weeks pre-pairing and up to eight weeks for females including a two-week pre-pairing phase, throughout pairing, gestation and until Day 13 of lactation.

Frequency of treatment:

Daily (Animals of the F1 generation were not dosed)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Positive control:

None

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the acclimatisation period, observations of the animals and their cages were recorded at least once per day. During dosing Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATIONS
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, therefore observations were made on these occasions without “blinding”.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males Weekly during acclimatisation. Before dosing on the day that treatment commenced (Day 1) and weekly thereafter. F0 females Weekly during acclimatisation. Before dosing on the day that treatment commenced (Day 1) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption for F0 animals was undertaken weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording as follows:
Days 0-6, 7-13 and 14-19 after mating.
Days 1-3, 4-6 and 7-12 of lactation.


WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (light general anesthesia induced by isoflurane)
- Collection vessel: collected into tubes containing EDTA as anticoagulant.
- Animals fasted: Not specified
- How many animals: The five lowest numbered surviving males per group. The first five surviving lactating females with a litter per group.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (light general anesthesia induced by isoflurane)
- Collection vessel: collected into tubes containing lithium heparin as anticoagulant.
- Animals fasted: Not specified
- How many animals: The five lowest numbered surviving males per group. The first five surviving lactating females with a litter per group.


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.
- Battery of functions tested: sensory activity and grip strength/ motor activity

IMMUNOLOGY: No


OTHER: THYROID HORMONE ANALYSIS (F0 Adult males and females)
- Time schedule: At Termination: All surviving F0 adult males and females (no samples were obtained from animals which failed to litter or with a total litter loss).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows for F0 animals:

Adrenals
Brain (including cerebrum, cerebellum and pons)
Cecum
Colon
Cowpers glands
Duodenum
Epididymides
Eyes
Glans penis
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
LABC (levator ani-bulbocavernosus) muscle
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - left axillary
- mesenteric
Ovaries
Peyer’s Patch
Prostate
Sciatic nerve
Seminal vesicles with coagulating glands
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical level)
Spleen
Sternum (with marrow)
Stomach
Testes
Thymus
Thyroid
Trachea
Urinary bladder
Uterus with cervix(weighed with oviducts)
Vagina

Statistics:

Statistical analyses were performed on the majority of data. For some parameters, including estrous cycles before treatment and stage of estrous cycle at termination, the similarity of the data was such that analysis was not considered to be necessary.

All statistical analyses were carried out separately for males and females. Food consumption before pairing was analysed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.

For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

On Day 9 of treatment, one female receiving 330 mg/kg/day was killed for welfare reasons.
On Day 23 of gestation, a second female receiving 330 mg/kg/day was killed for welfare reasons.
On Day 8 of lactation, a Group 4 female receiving 750 mg/kg/day was terminated early due to total litter loss.
On Day 9 of lactation, a third female receiving 330 mg/kg/day was terminated early due to total litter loss.
On Day 25 of gestation, three Group 4 females receiving 750 mg/kg/day were killed due to having surpassed the designated gestational time.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gains for males given 750 mg/kg/day were moderately low and slightly low for males given 100 or 330 mg/kg/day. During the two-week pre-pairing period the females receiving 750 mg/kg/day body weight gain was higher than Controls. During the gestation and lactation periods body weight gain was slightly or moderately low for females receiving 330 or 750 mg/kg/day. For females receiving 100 mg/kg/day, body weight gain was slightly low during the lactation period only.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food intake for males receiving 750 mg/kg/day was generally slightly lower compared to Control throughout the treatment period. Mean food consumption during gestation was low for females receiving 750 mg/kg/day and following parturition was low for females receiving 330 or 750 mg/kg/day. Mean food consumption for males receiving 100 or 330 mg/kg/day and for females receiving 100 or 330 mg/kg/day was generally similar to Controls throughout the dosing period, with exception of the lactation period for females at 330 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a reduction in red cell mass amongst males and females receiving 330 or 750 mg/kg/day. Mean cell haemoglobin and mean cell volume were slightly lower than that of Controls for males receiving 330 or 750 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry investigations after five weeks of treatment revealed statistically significant changes in the liver enzymes for males at all dose levels where alkaline phosphatase activity was higher compared to Controls and alanine aminotransferase activity was lower compared to Controls. A dose dependent response was observed for alkaline phosphatase only. Liver enzymes changes were observed in females with lower alkaline phosphatase and alanine aminotransferase activity at all dose levels compared to Controls. A dose dependent response was observed for alanine aminotransferase levels only. Electrolyte levels were high for males at 330 and 750 mg/kg/day when compared to Controls although there was no dose-response evident. Bilirubin and bile acids concentrations for males receiving 750 mg/kg/day were higher compared to Controls. Mean albumin concentrations were slightly high at all dose levels in males and in females receiving 750 mg/kg/day, a dose relationship was only apparent in males.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in organ weights consisted of slightly lower body weight adjusted seminal vesicles with coagulating gland and LABC weights in males receiving 750 mg/kg/day, adjusted prostate weights were also low in males receiving 330 or 750 mg/kg/day. In males, at all dose levels slightly high body weight adjusted kidney weights were seen. Slightly high body weight adjusted liver weights were evident amongst males at 330 or 750 mg/kg/day and in females given 330 mg/kg/day. In addition, group mean adjusted thymus weights were moderately high for females given 100, 330 or 750 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of the adult males and females revealed abnormally pale colouration of the livers in all males given 750 mg/kg/day, abnormally pale areas in the lungs of four males and six females given 750 mg/kg/day and two females receiving 330 mg/kg/day. Thickening of the non-glandular region of the stomach was also observed in two females receiving 750 mg/kg/day and one male receiving 330 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological evaluation of retained tissues revealed treatment related changes in the kidneys, lungs, stomach, mammary gland and liver. In the kidneys, vacuolation of the tubular epithelium was present in all males given 750 mg/kg/day and there was single cell necrosis of the tubular epithelium in three females given 330 mg/kg/day and all females given 750 mg/kg/day. Minimal to slight alveolar macrophage aggregates were distributed at the terminal bronchioles and/or perivascular within the subpleural region affecting all males and females given 750 mg/kg/day and in four females and two males receiving 330 mg/kg/day. This was accompanied by acicular clefting and neutrophilic infiltrate and associated type 2 epithelialisation (particularly for females at 750 mg/kg/day). Hyperplasia and/or hyperkeratosis were present in the mucosa of the non-glandular region of the stomach affecting a single male and female given 750 mg/kg/day and this was accompanied by minimal single erosion of the non-glandular mucosa at the limiting ridge in the male given 750 mg/kg/day; this was associated with gross thickening in some animals. The mammary tissue showed decreased secretory activity in four females receiving 750 mg/kg/day and one female given 330 mg/kg/day. Minimal to slight increase of rarefaction was present in the liver of all males given 330 or 750 mg/kg/day and the majority of females given 750 mg/kg/day and two females given 330 mg/kg/day. Centrilobular hypertrophy was observed in one male and in two females dosed with 750 mg/kg/day

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Circulating Thyroxine Levels: There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

330 mg/kg bw/day (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

330 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 100 mg/kg/day due to the histopathological changes in the kidneys and lungs.

Adverse findings included minimal increases of single cell necrosis of the tubular epithelium in females given 330 or 750 mg/kg/day andalveolar macrophages at the bronchioloalveolar junction with the associated inflammatory cell infiltrates of polymorphonuclear cells, acicular clefting and type 2 epithelialisation was observed in females and one male given 750 mg/kg/day. 
 

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test substance, KOMAD 710, following repeated dosing.  The assessment of repeated dose toxicity was made as part of a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test which also assessed potential adverse effects of the test item on reproduction. The study was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

 

Method

Three groups of ten male and ten female rats received the test substance at doses of 100, 330 or 750 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing and up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, propylene glycol,throughout the same relative treatment period.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

Results

Mortality

On Day 9 of treatment, one female receiving 330 mg/kg/day was killed for welfare reasons.

On Day 23 of gestation, a second female receiving 330 mg/kg/day (3F No. 96) was killed for welfare reasons.

On Day 8 of lactation, a Group 4 female receiving 750 mg/kg/day was terminated early due to total litter loss. 

On Day 9 of lactation, a third female receiving 330 mg/kg/day was terminated early due to total litter loss. 

On Day 25 of gestation, three Group 4 females receiving 750 mg/kg/day were killed due to having surpassed the designated gestational time.

Clinical Signs

There were no clinical signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment.

Behavioural Assessment

There were no treatment-related changes in the behavioural parameters observed during the study.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment.

Body Weight

Overall body weight gains for males given 750 mg/kg/day were moderately low and slightly low for males given 100 or 330 mg/kg/day. During the two-week pre-pairing period the females receiving 750 mg/kg/day body weight gain was higher than Controls. During the gestation and lactation periods body weight gain was slightly or moderately low for females receiving 330 or 750 mg/kg/day. For females receiving 100 mg/kg/day, body weight gain was slightly low during the lactation period, only.

Food Consumption

Mean food intake for males receiving 750 mg/kg/day was generally slightly lower compared to Control throughout the treatment period. Mean food consumption during gestation was low for females receiving 750 mg/kg/day and following parturition was low for females receiving 330 or 750 mg/kg/day. Mean food consumption for males receiving 100 or 330 mg/kg/day and for females receiving 100 or 330 mg/kg/day was generally similar to Controls throughout the dosing period, with exception of the lactation period for females at 330 mg/kg/day.

Haematology

The haematological examinations at scheduled termination revealed, when compared with Controls, a reduction in red cell mass amongst males and females receiving 330 or 750 mg/kg/day. Mean cell haemoglobin and mean cell volume were slightly lower than that of Controls for males receiving 330 or 750 mg/kg/day. 

Blood Chemistry

Blood chemistry investigations after five weeks of treatment revealed statistically significant changes in the liver enzymes for males at all dose levels where alkaline phosphatase activity was higher compared to Controls and alanine aminotransferase activity was lower compared to Controls. A dose dependent response was observed for alkaline phosphatase only. Liver enzymes changes were observed in females with lower alkaline phosphatase and alanine aminotransferase activity at all dose levels compared to Controls. A dose dependent response was observed for alanine aminotransferase levels only. Electrolyte levels were high for males at 330 and 750 mg/kg/day when compared to Controls although there was no dose-response evident. Bilirubin and bile acids concentrations for males receiving 750 mg/kg/day were higher compared to Controls. Mean albumin concentrations were slightly high at all dose levels in males and in females receiving 750 mg/kg/day, a dose relationship was only apparent in males. 

Organ Weights

Changes in organ weights consisted of slightly lower body weight adjusted seminal vesicles with coagulating gland and LABC weights in males receiving 750 mg/kg/day, adjusted prostate weights were also low in males receiving 330 or 750 mg/kg/day. In males, at all dose levels slightly high body weight adjusted kidney weights were seen. Slightly high body weight adjusted liver weights were evident amongst males at 330 or 750 mg/kg/day and in females given 330 mg/kg/day. In addition, group mean adjusted thymus weights were moderately high for females given 100, 330 or 750 mg/kg/day.

Necropsy

Macroscopic examination of the adult males and females revealed abnormally pale colouration of the livers in all males given 750 mg/kg/day, abnormally pale areas in the lungs of four males and six females given 750 mg/kg/day and two females receiving 330 mg/kg/day. Thickening of the non-glandular region of the stomach was also observed in two females receiving 750 mg/kg/day and one male receiving 330 mg/kg/day.  

Histopathology

Histopathological evaluation of retained tissues revealed treatment related changes in the kidneys, lungs, stomach, mammary gland and liver. In the kidneys, vacuolation of the tubular epithelium was present in all males given 750 mg/kg/day and there was single cell necrosis of the tubular epithelium in three females given 330 mg/kg/day and all females given 750 mg/kg/day. Minimal to slight alveolar macrophage aggregates were distributed at the terminal bronchioles and/or perivascular within the subpleural region affecting all males and females given 750 mg/kg/day and in four females and two males receiving 330 mg/kg/day. This was accompanied by acicular clefting and neutrophilic infiltrate and associated type 2 epithelialisation (particularly for females at750 mg/kg/day). Hyperplasia and/or hyperkeratosis were present in the mucosa of the non-glandular region of the stomach affecting a single male and female given 750 mg/kg/day and this was accompanied by minimal single erosion of the non-glandular mucosa at the limiting ridge in the male given 750 mg/kg/day; this was associated with gross thickening in some animals. The mammary tissue showed decreased secretory activity in four females receiving 750 mg/kg/day and one female given 330 mg/kg/day. Minimal to slight increase of rarefaction was present in the liver of all males given 330 or 750 mg/kg/day and the majority of females given 750 mg/kg/day and two females given 330 mg/kg/day. Centrilobular hypertrophy was observed in one male and in two females dosed with 750 mg/kg/day

Circulating Thyroxine Levels

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Conclusion

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 100 mg/kg/day due to the histopathological changes in the kidneys and lungs.

Adverse findings included minimal increases of single cell necrosis of the tubular epithelium in females given 330 or 750 mg/kg/day and alveolar macrophages at the bronchioloalveolar junction with the associated inflammatory cell infiltrates of polymorphonuclear cells, acicular clefting and type 2 epithelialisation was observed in females and one male given 750 mg/kg/day.