Glycerol monomyristate

Endpoint summary

Description of key information

Oral: OECD 422, rat, NOAEL ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 352-426 g (males; mean: 372 g), 192-249 g (females; mean: 224 g)
- Housing: individual in stainless steel cages
- Diet: ad libtum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 - 23.2
- Humidity (%): 48 - 61
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted in appropritate amounts of corn oil for each dose level. Aliquots of the dosing solution corresponding to the amount of daily administration were stored in the dark at 2 - 6 °C. The stability of the dosing solution was 7 days in a refrigerator and 1 day at room temperature. Therefore, the dosing solution was used within 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance showed low solubility in water.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): V4N3566

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details reported.

Duration of treatment / exposure:

males: 42 days (14 days prior to mating and 28 days thereafter)
females: 42-52 days (from 14 days before mating to day 4 of lactation)
satellite males and females: 42 days and 14 days post-exposure observation period

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females in control and test groups
7 males in control and 1000 mg/kg bw groups
12 males in 100 and 300 mg/kg bw groups
5 animals per sex in satellite control and 1000 mg/kg bw/day groups (in addition to number listed above)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Two dose range finding studies were performed. 2000 mg/kg bw of test substance was administered for 3 days in male and female rats. No abnormalities were found in general condition and body weight. The second study was performed at dose levels of 0, 330, 100, 300 and 1000 mg/kg bw/day for 14 days. No abnormalities of general condition, body weight, food consumption, hematological findings, blood biochemical findings, gross pathology and organ weight were found. Therefore, 1000 mg/kg bw/day was selected as the highest dose.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Day 1 (before administration), 7, 14, 21, 28, 35 and 42 (before sacrifice)
Females: Day 1 (before administration), 7, 14, during pregnancy on Day 0, 7 14 and 21, during lactation on Days 0 and 4 (before sacrifice)
Satellite males and females: Day 1 (before administration), 7, 14, 21, 28, 35, 42, 49 (Day 7 of recovery period) and 56 (Day 14 of recovery period, before sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last application
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelets, Reticulocytes, PT, APTT, WBC, Differential leukocytes: Lymphocytes, Neutrophils, Eosinophils, Basophils, Monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last application
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: AST, ALT, ALP, γ-GTP, T-protein, Albumin, A/G, T-bilirubin, BUN, Creatinine, Glucose, T-cholesterol, Triglyceride, Na, K, Cl, Ca, P, LDH, choline esterase

URINALYSIS: Yes (males)
- Time schedule for collection of urine: Day 37 during administration period or Day 9 during recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Colour, cloudiness, water consumed, volume, specific gravity, Na, K, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals, Fat

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: on Week 6 of the administration period
Females: on Week 6 of the administration period
Males and females of satellite groups: on Week 6 of the administration period and on Week 2 of the recovery period
- Dose groups that were examined: all
- Battery of functions tested: hearing reaction, eye sight reaction, sense of touch reaction, pain reaction, pupil reflex, pinna reflex, ipsilateral flexor reaction, eyelid reflex, righting reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Body surface, mucous membranes and internal organs

HISTOPATHOLOGY: Yes
Brain, pituitary, thyroid, thymus, lung trachea (after liquid immersion fixation), stomach, intestines, heart, liver, spleen, kidney, adrenal gland, bladder, testis, epididymis, prostate, seminal vesicles, ovaries, uterus, spinal cord (cervical, thoracic, lumbar), sciatic nerve, bone marrow (femur), lymph nodes (cervical lymph node, mesenteric lymph nodes), mammary gland, and other gross abnormalities
Spermatogenic cycle (Stage II, III, V, VII, and XII) was also investigated.

SACRIFICE
- Males: on Day 43
- Females: on Day 5 of lactation
- Females (unsuccessful mating): on Day 53
- Females (mated but non-pregnant): on Day 5 after scheduled delivery
- Satellite males and females: on Day 15 of the recovery period

Other examinations:

Organ weight: brain, liver, kidney, spleen, heart, thymus, thyroid, pituitary, adrenal, testis, sminal vesicle, epididymis
Estrous cycle
Number of ovarian corpora lutea
Number of uterine implantation
Observation of pups

Statistics:

ANOVA, Barlettm Kruskal-Willis, Dunnett, F test, Studen t-test, Aspin-Welch t-test, Mann-Whitney U-test, Fisher's exact test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, males): significant increase was observed (non adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, females): significant increase on Day 14 of administration (non adverse)

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):

100 and 300 mg/kg bw/day (males): decrease in APTT without dose-dependency after administration period (non adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day (females): significant increase in organic P in females after administration period without dose-dependency (non adverse); 1000 mg/kg bw/day (satellite group, females): significant increase in choline esterase (non adverse)

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: significant decrease in absolute weight of seminal vesicles in male and significant decrease in relative weight of spleen in females after administration period (non adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: mass in subcutis in one female after administration period (not treatment-related)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

control and 1000 mg/kg bw/day: after administration period, low incidence of commonly found microscopic changes, evenly distributed between groups (not treatment-related)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: after administration period, benign fibroadenoma of the mammary gland in one female (not treatment-related)

Details on results:

CLINICAL SIGNS AND MORTALITY
No abnormalities and no mortality were observed.

BODY WEIGHT AND WEIGHT GAIN
No change of body weight and weight gain was observed during the administration period. During the recovery period, a significant increase in body weight was noted in males at 1000 mg/kg bw/day. This was caused by a tendency of the control group animals to lose weight. One male in control group showed a significant decrease in body weight during the recovery period. However, no other abnormalities were observed in this male.

FOOD CONSUMPTION
No change was observed during the administration period in the test groups. A significant increase in food consumption was found in satellite females of the 1000 mg/kg bw/day group on Day 14 of administration. However, it was regarded as an incidental finding since the food consumption of the corresponding control group was relatively small on that day. Therefore, this change was not regarded as a compound-related effect.

HAEMATOLOGY
No changes were observed. After administration, a significant decrease in APTT was observed in males of the 100 and 300 mg/kg bw/day groups in comparison with control group. However, this change was not regarded as compound-related, because no dose-dependency was observed and this was within reference range.

CLINICAL CHEMISTRY
A significant decrease in inorganic P was observed in females of the 300 and 1000 mg/kg bw/day groups after administration. However, this change was not regarded as compound-related, since a dose-dependency was not observed and almost all the individual data were within the reference ranges except for one female of 300 mg/kg bw/day.
Choline esterase was increased in females of 1000 mg/kg bw/day after the recovery period but this was within the reference range. This change was not compound-related effect since this change was slightly and no change was observed after the administration period.

URINALYSIS
No significant changes were found during the administration and recovery periods.

NEUROBEHAVIOUR
No abnormality was observed for the sensory/reflex function, landing foot, grip strength and motor activity during the administration period. During the recovery period, motor activity increased from 0 to 60 min but not from 0 to 30 min in females dosed 1000 mg/kg bw/day. This effect was not compound-related since the observed change was slight and no change was observed during and after the administration period.

ORGAN WEIGHTS
After the administration period, a significant decrease in the absolute weight of seminal vesicles in males given and a significant decrease in relative weight of spleen in females were observed in the 100 mg/kg bw/day group. However, this change was not compound-related because no abnormalities were found at histopathological examination and there was no dose-dependency.
After the recovery period, a significant decrease in relative weight of pituitary in males of the 1000 mg/kg bw/day group and relative weight of thyroid in females of the 1000 mg/kg group bw/day was observed. Nevertheless, this effect was not compound-related because no abnormality was reported regarding these organs after administration period.

GROSS PATHOLOGY
No test substance-related changes were found. No abnormalities were found in breeding pairs which were not successful at mating and in those which were successful at mating but not pregnant.
After administration period, reddish thymus was noted in one male of the control group and subcutis mass was found in one female of 300 mg/kg by/day group (see also HISTOPATHOLOGY: NEOPLASTIC). After the recovery period, larger spleen and capsular thickening in spleen was found in one male and reddish area in thymus was observed in one female at 1000 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test substance-related changes were observed. There were also no changes regarding spermatogenic cycle.
Myocardial degeneration/fibrosis, foam cell accumulation in lung, mineralization in artery in lung, fatty degeneration of hepatocyte, microgranuloma in liver, solitary cyst in kidney, hyaline cast in kidney, lymphocyte infiltration in cortex of kidney, mineralization of cortico-medullary junction in kidney, fibrosis of cortex in kidney and haemorrhage in thymus were observed both in the control and 1000 mg/kg bw/day groups or only in the control group with low incidence. Hyaline droplet of proximal tubular epithelium in kidney was found in all males of the control and 1000 mg/kg bw/day groups. Brown deposit pigment and extramedullary haematopoiesis in spleen were found in all males and females of the control and 1000 mg/kg bw/day groups. However, there were no differences between control and test group. In the 1000 mg/kg bw/group, lymphocyte interstitium infiltration in prostate was found in one male, interstitial focal inflammation in lung and focal necrosis in liver was observed in one female. These changes occur naturally and were not compound-related.
No abnormalities were found in uterus and ovary in the non-pregnant females and the unsuccessful copulation females of the control and 1000 mg/kg bw/day groups, respectively. Degeneration of seminiferous tubules of testis, decrease in sperm and atrophy in prostate was observed in one control male.

HISTOPATHOLOGY: NEOPLASTIC
Mass on abdomen was found in a female after 40 days of administration in the 300 mg/kg bw/day group. However, this subcutaneous tumour of the mammary gland was a benign fibroadenoma and generated naturally.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Feb - 07 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

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

Species:

rat

Strain:

other: Crl:WI (Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277 - 353 g (males) and 183 - 225 g (females)
- Housing: 5 animals of the same sex per cage in Macrolon cages (MIV type). Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenised to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

DOSE VOLUME:
5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 h.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 5 females (main study)
5 males and 5 females (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 10-day dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males: Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.

Sacrifice and pathology:

DAY OF NERCROPSY:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanised in extremis.

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
From the first 5 Main animals/sex/group, all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):
Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

From all remaining animals and females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse)

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg bw/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg bw/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg bw/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg bw/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time in males at 1000 mg/kg bw/day and lower relative lymphocyte counts in females at 300 mg/kg bw/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg bw/day, and higher chloride levels in males at 100 mg/kg bw/day. Changes at 1000 mg/kg bw/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg bw/day had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg bw/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg bw/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg bw/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg bw/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg bw/day and the end of treatment, lower heart weight in females at 1000 mg/kg bw/day, higher heart to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg bw/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg bw/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discolouration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discolouration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg bw/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.

Key result

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable studies (Klimisch score 1) from reference substances with similar structure and intrinsic properties. Read-across is justified based on structural similarity and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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

Additional information

Justification for read-across

Data on the subacute repeated dose toxicity of Glycerol monomyristate (CAS 27214 -38 -6) are not available. The assessment of repeated dose toxicity was therefore based on data from source substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

The subacute oral toxicity of Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052 -13 -0) was investigated in Crl:WI (Han) rats according to OECD guideline 422 and in conformity with GLP (Riken, 2010). Dilutions of the test substance in polyethylene glycol were administered once daily to groups of 10 male and 5 female rats at doses of 100, 300 and 1000 mg/kg bw/day for 28 days via gavage. A similar constituted group received the vehicle and served as control. In addition, satellite groups of 5 males and 5 females each for the control and high dose group were used to investigate reversibility of effects during a 14-day post-exposure recovery period. No substance-related mortalities and clinical signs occurred during the whole study period. All parameters assessed at neurobehavioural examination were found to be normal and comparable to controls. Food consumption was similar between treated and control animals and no toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day. Any statistically significant changes in clinical biochemistry and haematology parameters were considered to be of no toxicological relevance as these occurred in the absence of a clear treatment-related trend and remained within the range considered normal for rats of this age and strain. Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a clear treatment-related trend. Further, organ weight changes were within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. In addition, no histopathological correlates were noted which could support organ weight changes. At necropsy, all incidental findings remained within the background range of findings that are encountered among rats of this age and strain. Since no dose-related trend was observed, these findings were considered to be of no toxicological relevance. Based on the results of this subacute toxicity study, the NOAEL for Crl:WI rats was considered to be ≥ 1000 mg/kg bw/day.

 

A GLP-compliant subacute oral toxicity study according to OECD 422 was performed with 2,3-dihydroxypropyl oleate (CAS 111 -03 -5) at doses of 100, 300 and 1000 mg/kg bw/day (MHLW, 2005). Male and female Sprague Dawley rats (12 per sex and group, except for 1000 mg/kg bw/day: only 7 males) received the test substance in corn oil once daily via gavage. A control group, consisting of 7 males and 12 females, was treated with the vehicle alone. The duration of treatment was 42 days (14 days prior to mating and 28 days thereafter) in males and 42-52 days (from 14 days before mating to day 4 of lactation) in females, respectively. Satellite groups of 5 animals per sex, each for the control and test groups, were used to investigate reversibility of effects during a 14-day post-exposure recovery period. No mortality and no clinical signs and effects on neurobehaviour were observed during the whole study period. No adverse effects on body weight development were observed during treatment and recovery period. The analysis of clinical, haematological and urinary parameters did not reveal any dose-dependent and toxicologically relevant changes in treated animals compared to controls. At 100 mg/kg bw/day, a significant decrease in absolute weight of seminal vesicles in males and a significant decrease in relative weight of spleen in females were observed at necropsy. Since these effects did not follow a dose-dependent relationship and were not accompanied by any histopathological changes in the respective organs, they were not considered to be toxicologically relevant. A decrease in relative weights of pituitary in males and thyroid in females was observed at 1000 mg/kg bw/day at the end of the recovery period. Since no abnormities were reported for these organs after the administration period, they were considered as not compound-related. No test substance-related changes were found at gross pathology. A low incidence of commonly found microscopic changes was observed, which was evenly distributed between all groups and therefore considered as incidental findings. Although one female showed a subcutaneous tumour of the mammary gland after 40 days exposure to 300 mg/kg bw/day, this benign fibroadenoma was considered to be generated naturally, since no effects were observed at the higher dose levels. Based on the overall effects observed in this subacute study, a NOAEL of 1000 mg/kg bw/day for male and female Sprague Dawley rats was derived.

Overall conclusion for repeated dose toxicity

There are no available studies on the repeated dose toxicity of Glycerol monomyristate (CAS 27214 -38 -6). Therefore analogue read-across from subacute studies from 2 source substances was applied. No toxicologically relevant effects were observed. The NOAEL values for repeated dose toxicity were at or above the currently applied limit dose of 1000 mg/kg bw/day. No hazard after repeated oral exposure was identified. Based on the available data and following the analogue approach, the target substance is not expected to have long-term toxic effects via the oral route.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that "substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Glycerol monomyristate, data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

The available data on repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.