Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No studies have been performed on the toxicity of dodecane-12-lactam to reproduction. Data from oral 90-day studies did not reveal any adverse effects on the reproductive organs in rats (NOAEL (rat): > 125 mg/kg bw), whilst in dogs an impairment of sperm maturation, decreased testes and ovary weights, and atrophy of the prostate was found at a severely toxic dose level (about 1,000 mg/kg bw/day) and was interpreted as secondary effect (NOAEL (dog) = 350 mg/kg bw d).

The substance ε-caprolactam, which is structurally related to dodecane-12 -lactam, was tested over three generations of rats for its potential reproduction toxicity by oral gavage of doses of 1000, 5000 and 10000 mg/kg diet. A decreased food consumption and bodyweight gain was observed on doses of 5000 and 10000 mg/kg diet in dams and offspring. At 10000 mg/kg diet macroscopic slightly damages of kidneys in male rats were observed. The birth behaviour of dams and the time of survival of offspring was not affected. The NOEL was determined as 1000 mg/kg diet (calculated: approx. 100 mg/kg bw/day) and the NOAEL (rat, ε-caprolactam) is >=5000 mg/kg diet (calculated: approx. >=500 mg/kg bw/day).

Furthermore a developmental toxicity study conducted with dodecane-12-lactam in rats showed no relevant toxic effects to reproduction indicating that the substance is not a reproductive toxicant (CIT, 2001).

Based on these studies it is concluded that effects on fertility of the substance dodecane-12-lactam (lauryl lactam) at doses, which do not cause parental toxicity, are rather unlikely. Therefore further studies regarding effects on fertility are not necessary for dodecane-12-lactam (lauryl lactam).

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction
Remarks:
other: subchronic toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1973-06-28 - 1973-10-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: limited documentation (test substance)
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Principles of method if other than guideline:
see Test Conditions
GLP compliance:
no
Limit test:
no
Species:
other: dog
Strain:
other: Beagle
Sex:
male/female
Details on test animals and environmental conditions:
- Suppliers:
Asta Werke (Austria): 18 dogs;
Graeflich Degenberg-Schonburgsches Rentamt (Germany): 7 dogs;
inhouse: 15 dogs
- Age: 26 weeks (mean)
- Weight at study initiation: males 11.8 kg, females 9.2 kg (mean)
Route of administration:
oral: feed
Vehicle:
other: feed or gelatine capsules
Details on exposure:
- Type of exposure: in the diet (low and mid dose), gelatine capsules (high dose)
- Vehicle: gelatine capsules (high dose), dispensed daily in 2 equally large doses in an interval of approx. 4.5 hours
- Concentration in food: 0.1 and 0.7 % (w/w) (low / mid dose)
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Exposure period: 13 weeks (low and high dose groups); 14 weeks (mid dose and control groups)
Post exposure period: none
Frequency of treatment:
6 days/week
Details on study schedule:
Number of generation studies: 0
Remarks:
Doses / Concentrations:
m: 44, 350, or 969 mg/kg bw/day; f: 49, 352, or 989 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
4 males + 4 females per group
Control animals:
yes
Positive control:
positive control substance caprolactam applied in food (7 % w/w) to one further group
Parental animals: Observations and examinations:
- Clinical signs: at least daily, during working time practically continuously
- Mortality: at least daily
- Body weight: at the beginning and weekly
- Food consumption: daily
- Ophthalmoscopic examination: before beginning and after 1.5 and 3 months
- Haematology:
Peripheral blood, before beginning and after 1.5 and 3 months: Counting of erythrocytes, reticulocytes, normoblasts, HEINZ' bodies, leucocytes, thrombocytes. Determination of hematocrit, hemoglobin, erythrocyte volume, leucocyte formula. Calculation of erythrocyte volume (MCV), hemoglobin content per erythrocyte (MCH), hemoglobin concentration in the erythrocyte (MCHC).
Bone marrow, at dissection: Cell formula, granulopoietic-erythropoietic quotient
- Biochemistry:
before beginning and after 1.5 and 3 months: Glucose, thromboplastin time, total protein and protein fractions, albumin-globulin quotient, sedimentation rate of erythrocytes, activity of GPT, GOT, AP; bilirubin, cholesterol, urea, creatinine, sodium, potassium
- Urinalysis:
before beginning and after 1.5 and 3 months: color quality and intensity, pH, albumin, glucose, ketone bodies, occult blood, bilirubin, urobilinogen, orgasmic and non-orgasmic sediment constituents.
- Liver function test: before beginning and after 1.5 and 3 months: 2-Dye-test according to Zimmer
- Kidney function test: before beginning and after 1.5 and 3 months: Phenol-red test
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
histological examinations of testis (seminiferous tubules, spermiogenesis, spermatozoons, Sertoli cells, Leyding cells, rete testis, inflammation, atrophy, hemorrhage, blood stasis, autolysis) and epididymis
Litter observations:
not applicable
Postmortem examinations (parental animals):
- Macroscopic: general condition, rigor mortis, hair coat, skin, eyes, nose, oral cavity, ears, preputium, vulva, anus, testes with epididymides, mammary glands, subcutaneous tissue - blood vessels and blood - and skeletal muscles, nerves, thyroid, parathyroid, abdominal cavity (peritoneum, contents), diaphragm, spleen mesentery lymph nodes, pancreas, mandibular gland, parotid gland, lymph nodes (retrophar., mandib., cerv. superf. and axillar.), tongue, pharynx, larynx, thoracic cavity (pleura, contents), thymus, trachea, lungs, mediastinum, pericardium, epicardium, heart, aorta, pulmonary artery, ureters, urinary bladder, urethra prostate, ovaries, oviducts, uterus, vagina, espohagus, stomach, small intestine, colon, caecum rectum, adrenals, kidneys, liver, gall bladder, hypophysis, brain
- Microscopic: heart, lungs, esophagus, stomach (large curvature, small curvature, pylorus region), small intestine (duodenum, jenunum, ileum), caecum, colon, liver, pancreas, kidney, urinary bladder, testes (right and left), epididymes (right and left), ovaries (right and left), prostate, uterus, thyroid with parathyroid, adrenals (right and left), cerebrum, cerebellum, periph. nerve, spleen, lymph nodes, bone marrow
Postmortem examinations (offspring):
not applicable
Statistics:
no data
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
LOW and MIDDLE dose groups: No effects on male or female genital organ weights (testes, prostate, seminal vesicles, ovaries).

The following observations are reported for the HIGH dosage group:
- Mortality and time to death: 1 female died after 5 weeks.
- Clinical signs: The general condition and behavior were severely affected. This was principally expressed as apathy, ataxia, sialorrhea, lateral positioning and tonoclonic spasms; the reaction to acoustic and visual stimuli was diminished.
- Body weight gain: The dogs lost an average of 25 % (male) or 20 % (female) of their initial body weight until the beginning of the 4th week. Until the end of the application period they gained weight again, but did not reach their initial body weights (final body weights of males -20 %, females -15 %).
- Food/water consumption: The food consumption was strongly reduced; a few dogs ate nothing for days. Food consumption (g/kg bw/day, mean 1st to 13th week, mean 1st to 14th week; n.d. = not determined):
Males:
Negative control: 43.9, 43.7;
low dose: 44.0, n.d.;
mid dose: 50.0; 50.0;
high dose: 31.3; n.d. (range: from 17.3 in week 2 to 42.6 in week 13);
Females:
Negative control: 42.4, 42.4;
low dose: 48.7, n.d.;
mid dose: 50.2; 50.3;
high dose: 38.4; n.d. (range: from 21.6 in week 3 to 50.0 in week 13);
From week 3 onwards additional food (200 g per animal and day) was offered to the high dose male and female animals; this food was completely eaten by females, whilst only 72% of it (144 g/day) were consumed by the male animals.
- Organ weights: A decrease was observed in the testes (-39 % absolute, -24 % relative), prostate (-67 % abolute, -58% relative), and ovaries (-50 % absolute, -39 % relative).
- Gross pathology: No indications of an effect
- Histopathology: The histological examination of the testes revealed an impairment of the sperm maturation; the prostate was atrophic. Atrophy was found in the uterus of the dog which died. Fibrosis of the endometrial stroma was observed in 1 dog.
Dose descriptor:
NOAEL
Effect level:
352 mg/kg bw/day
Sex:
female
Basis for effect level:
other: Various effects (for details see below) but no effects on reproductive organs
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day
Sex:
male
Basis for effect level:
other: various effects (for details see below) but no effects on reproductive organs
Dose descriptor:
LOAEL
Effect level:
969 mg/kg bw/day
Sex:
male
Basis for effect level:
other: impairment of sperm maturation
Dose descriptor:
LOAEL
Effect level:
989 mg/kg bw/day
Sex:
female
Basis for effect level:
other: impairment of sperm maturation
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
not applicable
Generation:
F1
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
no further relevant remarks
Conclusions:
Impairment of the sperm maturation and atrophy of the prostate at the highest exposure level (testes and prostate weights were also significantly decreased) is interpreted to be a secondary effect because this dose was clearly toxic to the animals as evidenced by a 25 % loss of body weight. Likewise the reduction in ovary weights at the highest dose should also be interpreted as a secondary effect, since the body weights of females were reduced. Histopathological changes of the ovaries were not detected. Thus for doses which are non-toxic to the animals, significant compund related adverse effects on reproductive organs could not be observed under conditions of this study.
Executive summary:

In a 90-day oral study with male and female beagle dogs (4 animals/dose/sex), administered with up to 989 mg/kg bw/day, the histological examination of the testes revealed an impairment of the sperm maturation and atrophy of the prostate at the highest exposure level (testes and prostate weights were also significantly decreased). This dose was clearly toxic to the animals as evidenced

by a 25 % loss of body weight, and the effect can therefore be interpreted as secondary. Likewise, the reduction in ovary weights at the highest dose (absolute: -50 %; relative: -39 %) should be interpreted as a secondary effect, since the body weights of females were reduced by 15 %. Histopathological changes of the ovaries were not detected.

Endpoint:
toxicity to reproduction
Remarks:
other: subchronic toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992-02-05 - 1992-06-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Supplier: Charles River France
- Age: about 7 weeks
- Weight at study initiation: 270 g (male) / 200 g (female)
Route of administration:
oral: gavage
Details on exposure:
- Vehicle: gum arabic solution supplemented with 0.5 % Tween 80
- Concentration in vehicle: 10 % (aqueous suspension)
- Total volume applied: 5 ml/kg bw
Details on mating procedure:
not applicable
Duration of treatment / exposure:
- Duration of test/exposure:
96 days for reversibility study; 90 days for other animals
- Post exposure period: days 97 - 126 (reversibility study)
Frequency of treatment:
7 days/week
Details on study schedule:
Number of generation studies: 0
Remarks:
Doses / Concentrations:
0, 5, 25, or 125 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 male and 20 female (low and mid-dose group);
25 male and 25 female (control and high dose group), 5 per sex and group of these in reversibility study
Control animals:
yes, concurrent vehicle
Positive control:
not applicable
Parental animals: Observations and examinations:
- Clinical signs: daily
- Mortality: daily
- Body weight: twice weekly
- Food consumption: weekly
- Ophthalmoscopic examination: days 83/84
- Haematology:
days 85/86: erythrocytes, hemoglobin, packed cell volume, Wintrobe's indices (mean corpuscular volume, hemoglobin, and hemoglobin concentration = MCV, MCH, MCHC), reticulocytes (percentage and abolute; only 10 last animals per sex and group), leukocytes, differential leukocyte count (percentage and absolute), thrombocytes.
coagulation day 97: prothrombin time, activated partial thromboplastin time, fibrinogen level
- Biochemistry:
days 90/91-121: glucose, urea, creatinine, total proteins, albumin, globulins, albumin/globulin ratio, triglycerides, cholesterols, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatases, gamma-glutamyl transpeptidase, sodium, potassium, chloride, calcium, inorganic phosphate
- Urinalysis:
first 5 animals/sex/group, days 45, 85: volume, pH, proteins, ketone bodies, bilirubins, urobilinogen, specific gravity, glucose, leukocytes, blood, nitrite, erythrocytes, leukocytes, epithelial cells, renal cells, casts, ammonium magnesium phosphate crystals, oxalate crystals, bacteria, parasites
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
not applicable
Postmortem examinations (parental animals):
- Macroscopic: skin and subcutaneous tissues, mammary tissue, liver, spleen, kidneys, thymus, heart, lungs, tracheobronchial lymph nodes, urinary bladder, ovaries, uterine tubes, uterine cervix, vagina, testes, epididymides, seminal vesicles, prostate, aorta, sciatic nerve, popliteal lymph nodes, femur and bone marrow, crural muscle, pancreas, esophagus, forestomach, glandular area (stomach), duodenum, jejunum, ileum, cecum, colon, rectum, mesenteric lymph nodes, salivary glands, thyroid glands, parathyroid glands, larynx, tongue, eyes, harderian glands, brain, pituitary, inner ear, femoral bone marrow
- Microscopic: same as macroscopic except for femur and bone marrow, tongue, and inner ear
Postmortem examinations (offspring):
not applicable
Statistics:
- parametric methods for statistical distributions; Levene test followed by 1-ANOVA and/or Student t test; Bonferroni's method or Scheffe's method for multiple comparisons
- non-parametric methods: Kruskall Wallis test
Reproductive indices:
no data
Offspring viability indices:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical abnormality was imputable to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
45F showed clinical, macroscopic and microscopic signs of esophagus perforation.
54 M showed microscopic signs of aspiration pneumonia.
153M and 170F showed no specific sign accounting for death (stress changes and agonal pulmonary edema). Ground glass appearance of centrilobular hepatocytes, although imputable to treatment, cannot be considered as a cause of death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No major difference was observed at any moment of the treatment or reversibility period between groups. The slight decrease in body weight observed in both sexes and in almost every group on days 86 and 91 was probably linked to the examinations performed at the end of treatment
(mainly fasting for blood sampling).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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):
No difference imputable to treatment was observed between groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The variations imputable to treatment were observed on D90 {end of treatment period) at the high dose level (125 mg/kg/d):
a slight increase in albumin and total protein levels in females only {39.3 g/l vs 37.3 g/l for albumin and 78.4 g/l vs 75.1 g/l for total proteins),
a moderate increase in potassium levels in males only ( 5.25 mM vs 4.74 mM).
On D121 (end of reversibility period), no statistically significant difference remained.
The following variations were not imputed to treatment:
the statistically significant increase in glucose levels observed on D90 in high-dose males was
too slight to be biologically significant (6.00 mM vs 5.55 mM), the increase in triglyceride levels observed on 0121 in the females of the control group (1.18 mM on 0121 vs 0.59 mM on 090) was linked to an isolated marked increase observed in one female (no. 28, 3.21 mM), the statistically significant decrease in total bilirubin levels observed on D90 in high-dose males and females was too slight to be biologically significant ( 1.5 µIM vs 1.9 µM in males, 2.0 µM vs 2.3 µM in females), the statistically significant decrease in ASA T levels observed on D90 in high-dose males has no biological significance (56 IU/l vs 66 IU/l),
the numerous statistically significant differences observed for sodium (D90 in high-dose males and females, D121 in males) and chlorides (D90 in treated males, D121 in males) were slight and not biologically significant.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The modifications attributed to treatment were the following:
moderate increase in potassium excretion in mid- and high-dose males (25 and 125 mg/kg/d) on D 45 (2728 and 2416 mc.moles vs 1774 me. moles),
moderate increase in sodium concentration and excretion in high-dose males (125 mg/kg/d) on D45 (40 mM vs 23mM and 802 mc.moles vs 370 mc.moles).
These changes were no longer observed on D85.
The following changes were not attributed to treatment because they were slight or not related to the dose:
slightly increased pH in low-dose females on D45, (7.7 vs 7.0) and decreased pH in mid-dose females on D85, (6.2 vs 7.7), increased specific gravity in low- and mid-dose females on D85 (1.025 and 1.027 vs 1.017).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No variation imputable to treatment was observed.
The following statistically significant variations were not attributed to treatment because they were slight and not biologically significant:
slight decrease in thymus absolute weight in mid-dose males on D97 (372 mg vs 445 mg),
slight increase in liver relative weight in low-dose females on D97 (33.653g/kg vs 31.169 g/kg),
slight increase in heart relative weight in high-dose females on D97 (3. 77 g/kg vs 3.58 g/kg),
slight decrease in pituitary relative weight in high-dose females on D97 (49.3 mg/kg vs 53.5 mg/kg).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The study of the macroscopic lesions observed and of their incidence did not reveal any lesion
imputable to treatment.
The main lesions observed in the animals found dead are the following:
45F Control group, found dead on D83.
Perforation of esophagus, mass under foreleg and large quantity of blood in the stomach.
54M Low-dose group (5 mg/kg/d), found dead on D35.
Marked general congestion of the lungs, congestion or dark discoloration of various
organs (adrenals, thymus, popliteal lymph nodes, brain).
153M High-dose group (125 mg/kg/d, found dead on D56.
Early autolysis, congestion of the lungs and presence of foam in the bronchi, congestion
of thymus, liver pale discoloration, meteorism.
170F High-dose group (125 mg/kg/d), found dead on D63.
Congestion of the lungs and presence of foam in the bronchi, congestion or dark
discoloration of various organs (adrenals, ovaries).
The cause of these deaths is discussed in section mortality.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Liver: The administration of LAUROLACTAM resulted in a modification of the appearance of the cytoplasm of centrilobular hepatocytes (ground glass appearance) in a few males from group 2
(25 mg/kg/d) and 3 (125 mg/kg/d).
Thyroid: an increase in the incidence of the columnar appearance (and vacuolar in a few cases) of the follicular epithelium was observed in animals from groups 2 and 3. It was not considered
positively attributable to the test compound, the variation being minor.

Animals nos.45F (Group 0- control), 54M (Group l - 5 mg/kg/d), 153M and l70F (Group 3- 125 mg/kg/d) dead during the study on days 83, 35, 56 and 63, respectively.
In addition to congestive changes of little significance, these animals presented:
animal no. 45F,
stress changes (chronic lymphoid atrophy of the spleen and thymus),
• changes giving evidence of gavage-induced trauma (purulent, granulomatous or fibrous
inflammatory cell infiltration of the esophagus, salivary glands and retromandibular
lymph nodes with presence of vegetal particles);
Death is probably secondary to these changes.

- animal no. 54M, edematous subacute aspiration pneumonia (with presence of vegetal
particles) and subacute or granulomatous inflammatory cell infiltration of the larynx and
trachea with hemorrhage of the larynx and tracheobronchial lymph nodes;
Death is probably secondary to pneumonia.

- animal no. 153M,
• ground glass appearance of centrilobular hepatocytes (see above),
• stress changes (adreno-cortical hyperplasia),
• slight changes of alveolar and perivascular pulmonary edema, probably agonal;
The cause of death could not be determined.

animal no. 170F,
• stress changes (lymphoid atrophy of the spleen),
• alveolar and perivascular pulmonary edema, probably agonal;
The cause of death could not be determined.


Other findings:

They are physiological or belong to the spontaneous pathology of the species and are reported in the summary tables, thyroid adenoma of animal no. 152M (Group 3 - 125 mg/kgfd) and leukemia of animal no. 110M (25 mg/kg/d) included.

Electron microscopy

The liver was not examined because no sample was taken from the animals presenting the hereabove-mentioned hepatic changes, for electron microscopic examination.
The examination of the kidney was not considered necessary.

In conclusion, the administration of LAUROLACTAM resulted in a ground glass appearance of centrilobular hepatocytes in a few males from the dose of 25 mg/kg/d upwards.

One Group 0 animal was found dead following a gavage-induced trauma; one Group I animal (5 mg/kg/d) died of aspiration pneumonia; two Group 3 animals (125 mg/kg/d) died presenting a few
stress changes and changes of pulmonary edema probably agonal, which could not account for death.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
- Mortality and time to death:
0 mg/kg: 1 female, day 83, esophagal perforation
5 mg/kg: 1 male, day 25, aspiration pneumonia
25 mg/kg: no mortalities
125 mg/kg 1 male, 1 female, days 56, 63, cause for mortality not obvious: Animals showed no specific signs accounting for death (stress changes and agonal pulmonary edema). Ground glass appearance of centrilobular hepatocytes (the female), although imputable to treatment, could not be considered as a cause.
- Food/water consumption: no abnormality detected
- Ophthalmoscopic examination: no abnormality detected
- Clinical chemistry: high dose group: moderate increase in potassium (males), no longer statistically significant by end of reversibility period (day 121); slight increase (females only) in total proteins (78.4 vs 75.1 g/l) and albumin (39.3 vs 37.3 g/l)
- Haematology: no abnormality detected
- Urinalysis:
day 45: moderate increase in potassium excretion of medium / high dose males (2728 / 2426 vs 1774 mc.moles);
moderate increase in sodium concentration and excretion of high dose males (40 vs 23 mM; 802 vs 370 mc.moles);
day 85: no abnormality detected
- Histopathology: ground glass appearance of centrilobular hepatocytes in 4 males each of mid and high dose groups

Target organ: liver
All observed effects returned to normal values after a recovery period of 30 days.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: effects on reproductive organs
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
not examined
Generation:
F1
Remarks on result:
not measured/tested
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
no
no further remarks
Conclusions:
In a 90-day study performed in accordance with OECD TG 408(1981), Sprague Dawley rats (20 animals/low and mid dose/sex; 25 animals/control and high dose/sex) were orally gavaged with dodecane-12-lactam at 0, 5, 25 and 125 mg/kg bw/day. Treatment with up to 125 mg/kg bw/day had no effects on male or female genital organ weights (testes, prostate, seminal vesicles, uterus, ovaries). Likewise, histopathology of these organs of the top dose group did not detect any significant effects attributable to treatment with the test compound.
Executive summary:

In a 90-day study performed in accordance with OECD TG 408(1981), Sprague Dawley rats (20 animals/low and mid dose/sex; 25 animals/control and high dose/sex) were orally gavaged with dodecane-12-lactam at 0, 5, 25 and 125 mg/kg bw/day. Treatment with up to 125 mg/kg bw/day had no effects on male or female genital organ weights (testes, prostate, seminal vesicles, uterus, ovaries). Likewise, histopathology of these organs of the top dose group did not detect any significant effects attributable to treatment with the test compound.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Studies on the toxicity of dodecane-12-lactam to reproduction have not been performed.

In a 90-day study performed in accordance with OECD TG 408(1981), Sprague Dawley rats (20 animals/low and mid dose/sex; 25 animals/control and high dose/sex) were orally gavaged with dodecane-12-lactam at 0, 5, 25 and 125 mg/kg bw/day. Treatment with up to 125 mg/kg bw/day had no effects on male or female genital organ weights (testes, prostate, seminal vesicles, uterus, ovaries). Likewise, histopathology of these organs of the top dose group did not detect any significant effects attributable to treatment with the test compound (Sanofi Recherche, 1993).

In a 90-day oral study with male and female beagle dogs (4 animals/dose/sex), administered with up to 989 mg/kg bw/day, the histological examination of the testes revealed an impairment of the sperm maturation and atrophy of the prostate at the highest exposure level (testes and prostate weights were also significantly decreased). This dose was clearly toxic to the animals as evidenced

by a 25 % loss of body weight, and the effect can therefore be interpreted as secondary. Likewise, the reduction in ovary weights at the highest dose (absolute: -50 %; relative: -39 %) should be interpreted as a secondary effect, since the body weights of females were reduced by 15 %. Histopathological changes of the ovaries were not detected (INBIFO, 1974; NOAEL = 350 mg/kg bw d).

The substance ε-caprolactam, which is structurally related to dodecane-12 -lactam, was tested over three generations of rats for its potential reproduction toxicity by oral gavage of doses of 1000, 5000 and 10000 mg/kg diet. A decreased food consumption and bodyweight gain was observed on doses of 5000 and 10000 mg/kg diet in dams and offspring. At 10000 mg/kg diet macroscopic slightly damages of kidneys in male rats were observed. The birth behaviour of dams and the time of survival of offspring was not affected. The NOEL was determined as 1000 mg/kg diet (calculated: approx. 100 mg/kg bw/day) and the NOAEL is 5000 mg/kg diet (calculated: approx.500 mg/kg bw/day) (MAK, 1990).

Furthermore a developmental toxicity study conducted with dodecane-12-lactam in rats showed no relevant toxic effects to reproduction indicating that the substance is not a reproductive toxicant (CIT, 2001).

Based on these studies it is concluded that effects on fertility of the substance dodecane-12-lactam (lauryl lactam) at doses, which do not cause parental toxicity, are rather unlikely. Therefore further studies regarding effects on fertility are not necessary for dodecane-12-lactam (lauryl lactam).


Effects on developmental toxicity

Description of key information

In a gavage study performed in accordance with OECD TG 414 (2001), dodecane-12-lactam had no adverse effects on the development of rats up to and including the highest tested dose level of 1,000 mg/kg bw/day. Doses of 250 and 1,000 mg/kg bw/day were maternally toxic as evidenced by reduced food consumption (-7 %/-11 %) and reduced body weight gain (-37 %/-50 %) and, at 1,000 mg/kg bw/day, poor general condition (NOAEL maternal toxicity: 50 mg/kg bw/day; NOAEL developmental toxicity: 1,000 mg/kg bw/day)

.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-04-25 - 2001-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2000, draft)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1998)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, L'Arbresle (France)
- Strain: Crl CD (SD) IGS BR
- Age: 10-11 weeks
- Weight at study initiation: 190-332 (mean 263) g
- Sex: females (sexually mature and primigravid)
- identification by CIT identity number via ear tattoo
Environmental conditions:
-acclimation period before treatment: 5 days
- room temperature: 22 °C (+/- 2°C)
-humidity: 50% (+/- 20%)
- light /dark cycle: 12h/12h (07:00-19:00)
-ventilation: about 12 cycles/hour of filtered, non-recycled air
-food: free access to A04 C pelleted maintenance diet (batch No. 10227; UAR, Villemoisson, Epinay-sur-Orge, France)
-water: free access to filtered (0.22µm filter) tap water
Route of administration:
oral: gavage
Vehicle:
other: mixture of gum Arabic (10%) and tween 80 (0.5%) in purified water
Details on exposure:
- Treatment: doses based on preliminary study (CIT/Study No. 20870 RSR)
- Concentration in vehicle: 10, 50, or 200 mg/ml (suspensions; homogeneity verified by analysis)
- Total volume applied: 5 ml/kg bw/day
- Type or preparation of particles: Daily. The test substance was ground to fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the desired concentrations, and then homogenized using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogenity: The lowest and the highest concentrations (10 and 200 mg/ml) were prepared under conditions representative of those of the study. From each dosage forms duplicate samples were taken at three different levels (top, middle, bottom) and analyzed by HPLC analysis (UV detection at 212 nm) for concentration of the test material to evaluate the homogenity.

Concentration: The concentration of samples taken from each doseage form (including the control) prepared for use on the first day of treatment and on the last day of treatment was determined by HPLC analysis (UV detection at 212 nm)
Details on mating procedure:
Females were mated at breeder's facilities. Mating was confirmed by detection of a vaginal plug (day 0 post-coitum). 5 days acclimatization period to the conditions of the study followed.
Duration of treatment / exposure:
day 6 through day 19 post-coitum inclusive
Frequency of treatment:
daily (once a day)
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 per group; only the first 20 pregnant females were taken into consideration for fetal examinations.
Control animals:
yes, concurrent vehicle
Details on study design:
no further relevant details
Maternal examinations:
- Body weight gain: days 2, 6, 9, 12, 15, 18, 20 post-coitum
- Food consumption: intervals days 2-6, 6-9, 9-12, 12-15, 15-18, 18-20 post-coitum
- Clinical observations (morbidity and mortality, clinical signs): at least once a day
- Hysterectomies: on day 20 post-coitum all females were killed. weight of gravid uterus was recorded for each pregnant female at hysterectomy (with at least one live fetus). The ovaries and uterus of females were examined. (see chapter: "Ovaries and uterine content")
- After hysterectomy, the females were subjected to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also performed.
Ovaries and uterine content:
number of corpora lutea; number and distribution of: dead and live fetuses, implantation sites (or uterine scars), early and late resorptions
Fetal examinations:
Examinations of fetuses were carried out only in the first 20 litters (femals with at least one live fetus): body weight, sex, external examination (all visible structures, surfaces and orifices); detailed examination of the soft tissue including all organs and structures of the head, neck, thorax and abdomen (according to Wilson technique, approximately on half of the fetuses per litter) and detailed examination of the skeleton (bone and cartilage of the head, spine, rib cage, pelvis and limbs) on the remaining fetuses per litter; sex of each live fetus was determined at the time of evisceration or at the time of serial sectioning; sex of each dead fetus was determined at the time of hysterectomy
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
Indices:
Data are expressed as group mean values +/- standard deviation or as proportions (wherever necessary, the experimental unit of comprison was the litter). Pre-implantation loss was calculated as follows: (number of corpora lutea - number of implantation sites/ number of corpora lutea)x100; Post-implantation loss was calculated as follows: (number of implantation sites-number of live fetuses/number of implantation sites)x100; Fetal findings were analyzed as follows: (number of fetuses with a particular finding/total number of fetuses examined) x 100
Historical control data:
reported for pre-implantation loss and fetal skeletal variation
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related clinical signs in the 50 and 250 mg/kg/day treated groups.
Severe clinical signs of poor clinical condition were recorded in a notable proportion of the animals given 1000 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no treatment-related death at the 50 and 250 mg/kg/day dose-levels.
At the 1000 mg/kg/day dose-level, two females which presented poor clinical condition were prematurely sacrificed on day 8 post-coitum.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight and body weight gain were similar in the control and the 50 mg!kg/day treated groups.
In the 250 and 1000 mg/kg/day, the body weight gain (gross or net) was moderately to markedly lower when compared to the control group over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was similar in the control and the 50 mg/kg/day treated group.
In the 250 and 1000 mg/kg/day, there was a significant slight to moderate effect on food consumption over the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic findings in the control and the 50 mg/kg/day treated groups.
In the 250 and 1000 mg/kg/day treated groups, except the findings recorded in the two prematurely killed females (yellowish deposit on the stomach), the few findings recorded were among those commonly reported in rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions or total resorptions in any group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed on the pre- or the post-implantation loss, or the sex ratio.
The minimally lower fetal weight recorded at 250 and 1000 mg/kg/day was considered to be secondary to the lower maternal body weight gain and thus, did not represent a direct adverse effect on the embryofetal development.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality and day of death: There was no mortality in the 0 (control), 50 and 250 mg/kg bw/day dose levels. At the 1000 mg/kg/day dose level, two females which presented poor clinical condition were prematurely sacrificed on day 8 post-coitum.
- Number aborting: no abortions in any group
- Number of resorptions: no total resorptions in any group
- Body weight: Slight to marked effects in mid- and high-dose groups:
50 mg/kg bw/day: gross 3 % above, net 2 % below control
250 mg/kg bw/day: gross 15 %, net 37 % below control
1000 mg/kg bw/day: gross 26 %, net 50 % below control
(gross = days 6 to 20; net = from day 6, corrected for weight of uterine content)
- Food/water consumption: Slight to marked effects in mid- and high-dose groups:
50 mg/kg bw/day: not affected
250 mg/kg bw/day: food consumption 7 % below control
1000 mg/kg bw/day: food consumption 11 % below control
- Description, severity, time of onset and duration of clinical signs:
50 mg/kg bw/day, 250 mg/kg bw/day: no clinical signs that were related to the treatment with the test substance
1000 mg/kg bw/day: Severe clinical signs of poor clinical condition in a notable proportion of the animals: piloerection, round back, sedation, dyspnea and/or hypokinesia in 14/24 females, generally from the beginning until the end of the treatment.
- Gross pathology incidence and severity: There was no macroscopic finding that was attributed to the treatment with the test substance in any group.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The minimally lower fetal weight recorded at 250 and 1000 mg/kg/day was considered to be secondary to the lower maternal body weight gain and thus, did not represent a direct adverse effect on the embryofetal development.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed on the pre- or the post-implantation loss, or the sex ratio.
The minimally lower fetal weight recorded at 250 and 1000 mg/kg/day was considered to be secondary to the lower maternal body weight gain and thus, did not represent a direct adverse effect on the embryofetal development.
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No fetal external malformations or variations that were ascribed to the treatment with the test substance were noted in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No fetal skeletal malformations or variations that were ascribed to the treatment with the test substance were recorded in any group.
Visceral malformations:
no effects observed
Description (incidence and severity):
No fetal soft tissue malformations or variations that were ascribed to the treatment with the test substance were recorded in any group.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter size and weights: No treatment-related effects were observed on the pre- and the post-implantation losses, and the sex ratio. The minimally lower fetal weight recoded at 250 and 1000 mg/kg/day was not statistically significant and considered to be secondary to the lower maternal body weight gain, and thus did not represent a direct adverse effect on the embryofetal development. Mean fetal weights:
50 mg/kg bw/day: 4.04 +/- 0.21 g
250 mg/kg bw/day: 3.89 +/- 0.30 g
1000 mg/kg bw/day: 3.87 +/- 0.26 g
control: 3.93 +/- 0.25 g
- Number of viable fetuses: no treatment-related effects
- Sex ratio: no treatment-related effect
- External abnormalities: No treatment-related fetal external malformations or variations were noted in any group.
- Soft tissue abnormalities: No fetal soft tissue malformations or variations that were ascribed to the treatment with the test substance were recorded in any group.
- Skeletal abnormalities: No fetal skeletal malformations or variations that were ascribed to the treatment with the test substance were recorded in any group. Findings were considered to be of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Key result
Developmental effects observed:
no
no further relevant remarks
Conclusions:
The test substance Lauryl lactam, which was administered orally in rats, was not maternotoxic at 50 mg/kg bw/day. At dose-levels of 250 and 1000 mg/kg bw/day, the test substance produced slight to marked maternotoxicity resulting in reduction in food consumption and body weight gain. In addition, some mortality and clinical signs of toxicity were recorded at 1000 mg/kg/day.
No embryo- and fetotoxicity was recorded at any dose-levels, and no teratogenic effects were found. Consequently, the No Observed Effect Level for maternal toxicity is 50 mg/kg/day and the No Observed Effect Level for embryofetal toxicity is 1000 mg/kg/day.
Executive summary:

The objective of this prenatal development toxicity study was to evaluate the potential toxic effects of the test substance Lauryl lactam on the pregnant female Sprague-Dawley rats an on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy: day 6 to day 19 post-coitum inclusive. Three groups of 24 mated female Sprague-Dawley rats, received the test substance Lauryl lactam by oral administration at 50, 250 and 1000 mg/kg/day from day 6 to day 19 post-coitum inclusive. A control group of 24 mated female was given the vehicle alone.

The test substance Lauryl lactam, which was administered orally in rats, was not maternotoxic at 50 mg/kg bw/day. At dose-levels of 250 and 1000 mg/kg bw/day, the test substance produced slight to marked maternotoxicity resulting in reduction in food consumption and body weight gain. In addition, some mortality and clinical signs of toxicity were recorded at 1000 mg/kg/day. No embryo- and fetotoxicity was recorded at any dose-levels, and no teratogenic effects were found. Consequently, the No Observed Effect Level for maternal toxicity is 50 mg/kg/day and the No Observed Effect Level for embryofetal toxicity is 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-03 to 2016-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
The test item was suspended in the vehicle to the appropriate concentration.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Species: Rabbit
- Source: Isoquimen, SL; Carretera de Sant Miquel del Fai, Km 3,5; 08182 Sant Feliu de Codines; Spain
- Strain: New Zealand White
- Age: 7 to 8.5 months
- body weight: 3.75 - 5.97 kg
- Diet: ad libitum, Commercial ssniff K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water: Tap water ad libitum
- Adaptation period: 61 days
- Identifikation: Each rabbit was uniquely identified by a tattooed continuous number between 1 and 96
-Housing: Except during the mating period, the dams were kept separately in breeding cages with wire floors
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.
Route of administration:
oral: gavage
Vehicle:
other: 0.8% aqueous hydroxypropylmethyl cellulose gel
Details on exposure:
ADMINISTRATION: 
- Frequency: once daily, day 6 to 28 of gestation
- Dose volume: 5 ml/kg b.w.
- Dose: 0, 100, 300, 1000 mg/kg/bw
- Animals: 96 females in groups 1 to 4, 24 females per group
Test item preparation
The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentration and was administered orally at a constant volume once daily from the 6th to the 28th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rabbits for mating remained untreated.
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC-UV method
Details on analytical verification of doses or concentrations:
At start of dosing:
Analysis of stability and concentration: Immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature. (3 samples / test item group; groups 2 - 4). Number of samples: 3 x 3 = 9
Homogeneity: At start of administration, during (middle) administration and before administration to the last animal of the dose group. (3 samples / test item group; groups 2 - 4). Number of samples: 3 x 3 = 9
At end of the dosing period:
Analysis of concentration: During treatment with the test item always before administration to the last animal of the group. (1 sample / test item group; groups 2 - 4). Number of samples: 1 x 3 = 3

Concentration and stability:
The results of the analysis of the test item concentrations in the test item formulations confirmed that the test item formulations were correctly prepared. The actual concentrations of the test item were within the expected range of 90.3% to 101.1% of the nominal concentrations. These results indicated correctly prepared test item vehicle mixtures, which were stable for at least 24 hours
Homogeneity:
The homogeneity of the formulation samples during the administration procedure was evaluated by comparing the concentration of samples which were taken at the start, during and before administration to the last animal with each other.
The analysis revealed a stable homogeneous test item-formulation for the low, interme-diate and high dose groups with a range of 91.2% to 103.7% (acceptable deviations ≤ 10%).

Details on mating procedure:
Sexually mature ('proved') male rabbits of the same breed served as partners.
Mating was monogamous. The mating partners were randomly chosen: 1 male and 1 female animal were placed in one cage in the forenoon. Successful copulation was ascertained by observation. The day of copulation was considered as day 0 of pregnancy. Only rabbits with positive evidence of copulation were included in the study. Rabbits lacking signs of coitus were excluded from the analysis and replaced by additional spare animals.
Duration of treatment / exposure:
From gestation day 6 until gestation day 28, laparotomy on gestation day 29
Frequency of treatment:
Once daily
Duration of test:
29 days
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsanguinated.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 female rabbits/dose , orally dosed with 0, 100, 300 or 1000 mg test item/kg b.w.
Evaluated litters: max 20 litters per group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The dose levels for the main study were selected in agreement with the Sponsor based on the results of a dose-range-finding study for a study of prenatal developmental toxicity in pregnant rabbits ( dated May 19, 2015). In this dose-range-finding study, New Zealand White Rabbits were treated with doses of 100, 300 or 1000 mg test item/kg b.w./day by oral gavage from the 6th to 28th day of pregnancy.
No noteworthy toxicity was noted for the dams. No influence on the embryo-fetal development was noted at any tested dose level.
Based on the results from the above dose-range-finding study, dose levels of 100, 300 or 1000 mg test item/kg b.w./day (administered by oral gavage from the 6th to 28th day of pregnancy) were selected in agreement with the Sponsor for the present study of prenatal developmental toxicity rabbits.
Maternal examinations:
Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the
group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating
to the individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
Animals were individually observed at least once daily for any signs of behavioural changes, reaction to treatment or illness.
Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms dis-appeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.


Viability
Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible.


Body weight
The weight of each rabbit was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight change was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29). Furthermore, the carcass weight and the net weight change from day 6 are given.

Carcass weight: Carcass weight = Terminal body weight - uterine weight
Net weight change from day 6 = Carcass weight - body weight on day 6

These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.


Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded daily. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.

The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in kg

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

EXAMINATIONS (NECROPSY), Examination of the dams
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsanguinated.
In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead.
After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations.

Ovaries and uterine content:
Evaluation / Parameters

Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions < 2 g, number and % per litter
- Late resorptions > 2 g, number and % per litter
- % litters with resorptions per group


Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group
- litter mean per sex and group

Weight of fetuses
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group
- litter mean per sex and group

Fetuses
- number and % per dam (alive)
- number and % per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- sex ratio per litter
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- mean % per sex and group


Runts
- number per dam
- mean per group

Malformed fetuses
- individual data per fetus
- type of malformation: number and incidence (%) per group and litter
- number of affected fetuses per group

Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100

Fetuses with variations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- Number of affected fetuses per group

Total variation rate [%] = fetuses per group with variations / fetuses per group x 100


Fetuses with retardations
- individual data per fetus
- type of retardation: number and incidence (%) per group and litter
- number of affected fetuses per group11
Total retardation rate [%] = fetuses per group with retardations / fetuses per group x 100


Pre-implantation loss (%)
- per dam
- mean per dam
- per group

Pre-implantation loss [%] = corpora lutea - implantations / corpora lutea x 100

Post-implantation loss ( % )
- per dam
- mean per dam
- per group


Post-implantation loss [%] = implantations - living fetuses / implantations x 100



Fetal examinations:
Examination of the fetuses
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(h) The fetuses were sacrificed by an ether atmosphere.

Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages.
The head was removed from 50% of the fetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head struc-tures (e.g. eyes).
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
- Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or
non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA
yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data :
- The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)
- The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy) or an
internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).

The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses
per group was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.
Historical control data:
LPT Background Data: Values of control groups (n = 9) and test groups (n = 28); data taken from the years 2013 to 2015
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related changes of behaviour or external appearance were noted in the dams after oral treatment with 100, 300 or 1000 mg Test item/kg b.w./day.
Mortality:
no mortality observed
Description (incidence):
No test item-related premature death was noted in any of the test item-treated groups (100, 300 or 1000 mg Test item/kg b.w./day).
One intermediate dose dam (no. 54) was sacrificed prematurely on gestation day 19 due to abortus incipiens.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related adverse changes in body weight or body weight gain were noted in the dams after oral treatment with 100, 300 or 1000 mg Test item/kg b.w./day.

A slight but statistically significant (at p ≤0.05) reduction in body weight gain compared to the control was noted for the high dose dams (1000 mg Test item/kg b.w./day) on gestation days 6 to 9. This transient reduction was obviously a result of distinctly reduced food intake during this time period and is not considered to be adverse due to the short-lasting time interval.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 100 or 300 mg Test item/kg b.w./day. The slight but statistically significant reduction (at p y< 0.01 compared to the control group) noted on gestation days 9 to 10 for the food intake of the low dose group is considered as spontaneous and not test item-related. No corresponding reduction was noted for the intermediate dose group.
Treatment with 1000 mg Test item/kg b.w./day caused transient reductions in the food intake from start of treatment onwards. The maximum reduction in food consump-tion was 53.1% below the value of the control group (significant at p < 0.01) noted on gestation days 6 to 7. The food consumption of the high dose dams recovered after a few days and reached again the value of the control group on gestation day 17 to 18. Statistically significant reductions of the mean food consumption compared to the control (at p < 0.05 or p < 0.01) were noted on gestation days 6 to 7, 8 to 9, 9 to 10, 10 to 11, 14 to 15 and 15 to 16.
This transiently reduced food consumption observed predominantly during the first treatment days at the high dose level was reversible and therefore regarded to be not adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water were noted for the dams treated with 100, 300 or 1000 mg Test item/kg b.w./day by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Uterus weight and carcass weights:
No test item-related changes in the gravid uterus weight and the carcass weight (terminal body weight minus gravid uterine weight) in comparison to the control group were noted for the dams of the low, intermediate dose or high dose group (100, 300 or 1000 mg Test item/kg b.w./day).
Body weight change from day 6
No test item-related changes between the control group and the treatment groups (100, 300 or 1000 mg Test item/kg b.w./day) were noted for the absolute body weight gain and the net body weight gain (without gravid uterus) between gestation day 6 and gestation day 29
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy revealed no test item-related findings at 100 mg Test item/kg b.w./day.
The following hepatic changes were noted in a few intermediate and high dose dams as a result of increased work load and hence, are considered not to be test item-related:
At 300 mg Test item/kg b.w./day, paleness of the liver was noted in 4 of 17 terminally sacrificed dams (nos. 50, 51, 53 and 69). Further hepatic changes (increased lobular pattern and multiple yellow foci) were noted in the aborting intermediate dose dam (no. 54).
At 1000 mg Test item/kg b.w./day, hepatic changes (liver: paleness, partly thickened, porous or yellow discoloured apex) were noted in 5 of 19 terminally sacrificed dams (nos. 73, 74, 78, 86 and 95).
The following macroscopical changes in individual dams are considered to be of spontaneous nature:
- Markedly inflated intestines were noted in the low dose dam no. 29.
- Displaced left kidney was noted in the low dose dam no. 43.
- Paleness of placentae was noted in the high dose dam no. 78.
- A clear cyst was noted on the left kidney of the high dose dam no. 90.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg Test item/kg b.w./day, one dam (no. 54) with abortus incipiens was noted on gestation day 19 and excluded from reproductive evaluation. As an abortion is known to occur spontaneously in rabbits of this strain and age, this single incidence is considered to be an incidental finding and hence, is judged as not test item-related.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Total post implantation loss
At 100 mg Test item/kg b.w./day, a total post-implantation loss of 9 early resorptions was noted in dam no. 43. This finding is known to occur spontaneously in rabbits of this strain and age. The incidence of one total post-implantation loss is considered as spontaneous and is judged as not test item-related above all as no complete loss of litter was noted at the higher tested dose levels of 300 or 1000 mg Test item/kg b.w./day.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
see above
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
In total, 6 dead fetuses were found at laparotomy: 5 dead fetuses (including one runt) were noted in the litters of two low dose dams treated orally with 100 mg Test item/kg b.w./day and one dead runt was noted in the control group.
In detail, four dead fetuses (no. 1 f (runt), 2 m, 3 m and 4 f) were noted in the litter of the low dose dam no. 29 and one dead fetus (no. 10 m) in the litter of the low dose dam no. 38. The incidence of 5 dead fetuses in the low dose group is considered to be within the spontaneous variability and not test item-related as 4 of 5 dead fetuses were found in one litter. Above all, no dead fetuses were noted in the litters of the intermediate and high dose dams (treated orally with 300 or 1000 mg Test item/kg b.w./day).
The dead control fetus (11 m, runt) was found in the litter of dam no. 18.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Weight of placentae
The mean placental weights in test groups 2, 3 and 4 (dams treated with 100, 300 or 1000 mg Test item/kg b.w./day) were comparable to that of the control fetuses. All placental weights were within the normal variability. Slight differences observed in comparison to the control are without any biological relevance.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the mean fetal weights after administration of 100, 300 or 1000 mg Test item/kg b.w./day.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No test item-related influence was noted on the mean fetal weights after administration of 100, 300 or 1000 mg Test item/kg b.w./day.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Prenatal fetal development
No test item-related influence on the number of resorptions and the number of live fetuses were noted for the low and intermediate dose dams (100 or 300 mg Test item/kg b.w./day).
An increased post-implantation loss of 13.6% (significant at p ≤ 0.01, Chi2 test based on the group level) was noted at 100 mg Test item/kg b.w./day when compared to the incidentally low post-implantation loss of 2.7% of the control group. This finding (includ-ing the total post-implantation loss of one litter) is considered to be within the spontane-ous variability, above all, as no corresponding findings were noted at 300 mg Test item/kg b.w./day.
At 1000 mg Test item/kg b.w./day, increases compared to the control were noted for the post-implantation loss of 15.4% (significant at p ≤ 0.01, Chi2 test based on the group level) and for the incidence of total resorptions (significant at p ≤ 0.05, Dunnett test). However, all findings were within the range of LPT background data. The afore-mentioned findings are judged as not test item-related but within the spontaneous variability.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 2, 3 and 4 (100, 300 or 1000 mg Test item/kg b.w./day) was comparable to the control fetuses. Slight differences observed in the sex distribution in comparison to the control are without any biological relevance.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopically visible gross alterations (malformations or variations) were noted for the fetuses of the test item-treated groups (100, 300 or 1000 mg Test item/kg b.w./day) during the external examination of the fetuses.
Spontaneous alterations were noted in two fetuses of the control group in the form of external malformations of the head (acephalostomia or anencephaly) accompanied by external variations of the extremities (hyperflexion of the fore paws) in one of them.

A macroscopic internal examination was performed to detect gross alterations of the internal organs. No malformations or variations were noted during the internal examination of the fetuses of the control group and the treatment groups (100, 300 or 1000 mg Test item/kg b.w./day).
The external inspection of the brain in 50% of the fetuses revealed no changes for any of the fetuses after opening of the cranium and removal of the brain.

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations
No alterations in the form of malformations were noted during skeletal examinations of the fetuses according to DAWSON at any tested dose level (100, 300 or 1000 mg Test item/kg b.w./day).
The external malformation of the head (anencephaly) noted in one control fetus (dam no. 18, fetus 9 f) was confirmed by skeletal findings in the form of a distinctly enlarged fontanelle (approx. 10 x 5 mm) and unossified areas of os parietal (diameter approx. 3 mm), os interparietal (diameter approx. 2 mm) and os supraoccipital (diameter approx. 4 mm).
A skeletal examination of the head could not be performed for the second malformed control fetus (dam no. 11, 9 f) due to the congenital absence of the head (acephalosto-mia). Examination of the further skeleton revealed no malformations in this fetus.

Skeletal variations
The skeletal variations observed during examination according to DAWSON were related to the skull (fontanelle enlarged), the sternum (sternebra(e) bipartite, dumbbell-shaped, misaligned, misshapen and/or fused to a slight degree or supernumerary sternebra(e), the thoracic vertebral bodies (misaligned, misshapen and/or fused, supernumerary thoracic vertebral body), the caudal vertebral bodies (misaligned) and the rib(s) (accessory 13th ribs (uni- or bilateral), fused, misshapen, short or thickened rib).
No test item-related skeletal variations were noted at the low and intermediate dose level (100 or 300 mg Test item/kg b.w./day). Mostly only one or few fetuses were affected. The fetal incidences of the observed skeletal variations are within the LPT background range or showed no dose-response relationship (rib(s) short, see table on the next page).
At 1000 mg Test item/kg b.w./day) statistically significant increases compared to the control (at p ≤ 0.05 or p ≤ 0.01) were noted for the fetal incidences of variations of the ribs (accessory 13th ribs), the sternum (sternebra(e) bipartite) and for total skeletal variations. These findings are classified to be not adverse and could be explained as secondary effects due to the maternal stress caused by materno-toxic effects, e.g. reduced food consumption. Such an effect on skeletal variations is well-known .

Skeletal retardations
The retardations observed during skeletal examination (according to DAWSON) were related to the sternum (sternebra(e) unossified, incompletely ossified or reduced in size), the thoracic vertebral bodies (dumbbell-shaped or reduced in size), the lumbar vertebral arches (unossified) or the talus (unossified).
No test item-related influence was noted for the incidence of skeletal retardations at 100, 300 or 1000 mg Test item/kg b.w./day.
The statistically significantly increased incidences for skeletal retardations of the sternum and total fetal skeletal retardations (at p ≤ 0.05 or p ≤ 0.01) are well within the range of LPT background data and the significance resulted from incidentally low fetal incidences in the control and/or low and intermediate dose groups. Hence, the following statistically significant differences compared to the control are not considered to be test item-related.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related visceral alterations in the form of malformations were noted during soft tissue examinations of the fetal head according to WILSON at any tested dose level (100, 300 or 1000 mg Test item/kg b.w./day).
No test item-related influence was noted in the number of soft tissue variations of the fetal head compared to the control at any tested dose level (100, 300 or 1000 mg Test item/kg b.w./day).
Classified soft tissue variations of the fetal head (according to WILSON) revealed dilatation of the 4th cerebral ventricle and (subdural) haemorrhages/haematoma in the meninges, the cerebrum and the cranium in individual fetuses at all tested dose levels and/or in the control group.
The observed cystic areas in the cerebral hemisphere or the cerebrum noted in altogether 3 fetuses of the test item groups and in one control fetus are known to be fixation-induced artefacts .
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effect was noted for the number of runts. In total, 10 runts were noted at laparotomy: four runts were found at 100 mg Test item/kg b.w./day, one runt at 300 mg Test item/kg b.w./day and and two runts at 1000 mg Test item/kg b.w./day.
Three runts were found in the control group, one of them exposing external alterations (acephalostomia, hyperflextion of fore paws).
All afore-mentioned incidences are within the spontaneous range of variability and in the range of LPT background data.
Details on embryotoxic / teratogenic effects:
ABSTRACT and ASSESSMENT of fetal alterations
At 300 mg Test item/kg b.w./day, one dam with abortus incipiens was noted on gestation day 19 and excluded from reproductive evaluation. As an abortion is known to occur spontaneously in rabbits of this strain and age, this single incidence is considered to be an incidental finding and hence, is judged as not test item-related.
In total, 6 dead fetuses were found at laparotomy: 5 dead fetuses (including one runt) were noted in the litters of two low dose dams treated orally with 100 mg Test item/kg b.w./day and one dead runt was noted in the control group. No dead fetuses were noted in the litters of the intermediate and high dose dams (treated orally with 300 or 1000 mg Test item/kg b.w./day). No test item-related increase was noted for the incidence of runts at any tested dose level.
At 100 or 300 mg Test item/kg b.w./day, no test item-related alterations (malfor-mations or variations) were noted during the macroscopic external examination and the macroscopic gross inspection of the internal organs at laparotomy and the skeletal examination according to DAWSON or the soft tissue examination of the fetal heads according to WILSON.
At 1000 mg Test item/kg b.w./day, test item-related increased fetal incidences were noted for variations in skeletal ossification of the ribs (accessory 13th ribs), the sternum (sternebra(e) bipartite) and for total skeletal variations. These findings are classified to be not adverse and could be explained as secondary effects due to the maternal stress caused by materno-toxic effects, e.g. reduced food consumption. Such an effect on skeletal variations is well-known .
All further prenatal changes noted at 100, 300 or 1000 mg Test item/kg b.w./day are without any biological relevance.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
Developmental effects observed:
no

Examination of the dams

Summary of animals examined

Test item

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Treated females

24

24

24

24

Not examined females

(excluded/spare animals)

none

1#

none

none

Non-pregnant females

7

2

6

5

Evaluated pregnant females

17

21

18

19

Dams without viable fetuses

(total post implantation loss)

none

1

none

none

Dams with abortion

none

none

1

none

Evaluated litters with

viable fetuses

17

20

17

19

 

#:

Dam no. 35 died after misgavage.

Summary of animals examined

Test item

dose in mg/kg

b.w./day p.o.

Animal nos.

of mated

rabbits

Litters with viable fetues

(animal nos.)

Dams with abortion

(animal nos.)

Dams not

pregnant (NP)

or without viable fetuses (NVF)

(animal nos.)

Spare

animals, not

examined

(animal nos.)

Group 1:

Control

1 - 24

2-7, 10-13,

16-18, 20-23

none

NP: 1, 8, 9, 14, 15, 19, 24

none

Group 2:

100

25 - 48

25-30, 32-34, 36-42, 45-48

none

NP: 31, 44

NVF: 43

35#

Group 3:

300

49 - 72

49-53, 55-59, 62-65, 67, 69, 71

54

NP: 60, 61, 66, 68, 70, 72

none

Group 4:

1000

73 - 96

73-75, 77, 78, 81, 82, 84-95

none

NP: 76, 79, 80, 83, 96

none

#:

Premature death after misgavage.

Body weight and body weight gain

Body weight gain

(mean %)#

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Gestation day 0 to

gestation day 29

11.6%

8.7%

11.1%

8.6%

Gestation day 6 to

gestation day 29

9.7%

8.4%

11.2%

8.3%

Necropsy findings  

Group

Dam No.

Fate

Macroscopic finding

1

Control

 

 

No pathological findings

2

100 mg

test item/kg

29

Laparotomy

(GD 29)

Intestines: inflated (marked)

43

(NVF)

Laparotomy

(GD 29)

Kidney (left): displaced

3

300 mg

test item/kg

50

Laparotomy

(GD 29)

Liver: paleness

51

Laparotomy

(GD 29)

Liver: paleness

53

Laparotomy

(GD 29)

Liver: paleness

54

Abortus incipiens

(GD 18/19)

Liver: increased lobular pattern,

multiple yellow foci

(diameter approx. 1 to 3 mm)

69

Laparotomy

(GD 29)

Liver: paleness

4

1000 mg

test item/kg

73

Laparotomy

(GD 29)

Liver: paleness, partly thickened

74

Laparotomy

(GD 29)

Liver: porous, paleness,

yellow discoloured apex

78

Laparotomy

(GD 29)

Liver: paleness, partly severely pale

Placentae: paleness

86

Laparotomy

(GD 29)

Liver: yellow discoloured apex

(approx. 40 x 10 mm)

90

Laparotomy

(GD 29)

Kidney (left): clear cyst

(diameter approx. 2 mm)

95

Laparotomy (GD 29)

Liver: paleness of border

Conclusions:
In conclusion, under the present test conditions, the no-observed-adverse effect level (NOAEL) was above 1000 mg Test item/kg b.w./day for the dams. The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 1000 mg Test item/kg b.w./day. No test item-related effects were noted on the fetus.
Executive summary:

Aim of the study was the examination of the influence of the test item on the pregnant rabbit and the fetus when administered orally during the critical period of organogenesis and the fetal development (6th to 28th day of gestation).

In this prenatal developmental toxicity study, the test item was administered orally to female rabbits at dose levels of 100, 300 or 1000 mg/kg b.w./day from the 6th to 28th day of pregnancy.

Findings:

Examination of the dams

 

Mortality

No premature death was noted in the treatment groups (100, 300 or 1000 mg Test item/kg b.w./day) or in the control group.

One intermediate dose dam was sacrificed prematurely on gestation day 19 due to abortus incipiens.

Clinical signs

No test item-related effects were noted.

Body weight and

body weight gain

 

No test item-related influence was noted on body weight and body weight gain.

Food consumption

Treatment with 1000 mg Test item/kg b.w./day caused transient reductions in the food intake predominantly during the first treatment days. The maximum reduction in food consumption was 53.1% below the value of the control group (significant at p £ 0.01) noted on gestation days 6 to 7. Statistically significant reductions of the mean food consumption compared to the control (at p < 0.05 or p < 0.01) were noted on gestation days 6 to 7, 8 to 9, 9 to 10, 10 to 11, 14 to 15 and 15 to 16.

Drinking water consumption

No test item-related influence was noted for the drinking water consumption at any of the tested dose levels (visual assessment).

Necropsy findings

At 300 and 1000 mg Test item/kg b.w./day, necropsy revealed macroscopic hepatic changes (pale or yellowish areas) in 5 of 18 dams of the intermediate dose group (including the aborting dam) and in 5 of 19 dams of the high dose group. This finding was obviously due to increased work load of the liver at the high dose levels and not considered as test item-related.

Uterus and carcass weights

No test item-related influence was noted for the gravid uterus weight or the carcass weight (without uterus).

 

 

Examination of the fetuses

No test item-related influence was detected on the prenatal fetal development at 100, 300 or 1000 mg Test item/kg b.w./day with respect to the incidence of resorptions, number of live fetuses and the values calculated for the post-implantation loss.

 

 

 

No test item-related increase was noted for the incidence of dead fetuses or runts in the test item groups in comparison to the control group.

 

 


 

Sex distribution

 

No test item-related differences were noted.

 

Fetal weights

 

No test item-related differences were noted.

 

Placental weights

 

No test item-related differences were noted.

 

 

Fetal alterations

 

Malformations

No test item-related malformation was noted during the external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON.

 

 

Variations

The external examination at laparotomy, the gross inspection of the internal organs and the soft tissue examination of the fetal head according to WILSON revealed no test item-related variations.

At 1000 mg Test item/kg b.w./day, the skeletal examination according to DAWSON revealed statistically significantly increased incidences compared to the control (at p ≤ 0.05 or p ≤ 0.01) for variations of the ribs (accessory 13th ribs), the sternum (sternebra(e) bipartite) and for total skeletal variations.

 

 

Retardations

Examination according to DAWSON revealed no test item-related increased incidences of skeletal retardations.

 

 

Analysis of test item

formulations

 

The measured actual concentrations of the test item in the test item vehicle mixtures were between 90.3% and 103.7% of the nominal concentrations, indicating correctly prepared and homogenized formulations which were stable at room temperature for at least 24h.

Under the present test conditions, the no-observed-adverse effect level (NOAEL) was above 1000 mg Test item/kg b.w./day for the dams.

The transiently reduced food consumption observed predominantly during the first treatment days at the high dose level was reversible and therefore regarded to be not adverse.

Necropsy revealed macroscopic hepatic changes in a few intermediate and high dose dams (300 and 1000 mg Test item/kg b.w./day) which were obviously due to increased work load of the liver and considered asnottest item-related.

The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 1000 mg Test item/kg b.w./day. No test item-related effects were noted on the fetus.

However, at 1000 mg Test item/kg b.w./day, increased fetal incidences were noted for variations of the ribs (accessory) and the sternum (bipartite) during skeletal examination according to DAWSON.These findings are classified to be not adverse and could be explained as secondary effects due to the maternal stress caused by materno-toxic effects, e.g. reduced food consumption. Such an effect on skeletal variations is well-known.

No test item-related influence was noted on the reproductive parameters (number of implantation sites, number of resorptions and number of fetuses). No test item-related increase was noted for the number of dead fetuses.

No malformations were noted in the test item-treated groups. No test item-related increases in the fetal incidence of variations were noted during the macroscopic external or internal inspections at laparotomy, or soft tissue examination of the fetal head (according to WILSON). Skeletal examination according to DAWSON revealed no test item-related increase of skeletal retardations.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
other: rat and rabbit
Quality of whole database:
Klimisch 1 (reliable without restrictions)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a study performed in accordance with OECD TG 414 (2001), pregnant Sprague Dawley rats were gavaged with doses of 50, 250 or 1,000 mg/kg bw/day on days 6 through 19 post-coitum inclusive (CIT, 2001). 

In dams, slight to marked effects on food consumption and body weight gain were recorded in the 250 and 1000 mg/kg bw/day groups:

Influence on:

Dose level (mg/kg bw/day)

50

250

1000

Food consumption (compared to control)

±0 %

-7 %

-11 %

Net body weight gain* (compared to control)

-2 %

-37 %

-50 %

·         net body weight gain = increase in body weight after day 6, corrected for weight of uterine content

 

Clinical signs of poor general condition were only recorded in a notable proportion of the animals given 1000 mg/kg/day (resulting in the premature sacrifice of 2 animals). The NOAEL for maternal toxicity was 50 mg/kg bw/day.

Test substance-related adverse effects on the fetuses were not observed in any of the treatment groups.Parameters evaluated were body weight, pre- or post-implantation loss, sex ratio, external/skeletal/soft tissue malformations and variations.

In a second prenatal developmental toxicity study according to OECD 414 (LPT, 2016), the test item was administered orally to female rabbits at dose levels of 100, 300 or 1000 mg/kg b.w./day from the 6th to 28th day of pregnancy.

Under the present test conditions, the no-observed-adverse effect level (NOAEL) was above 1000 mg Test item/kg b.w./day for the dams.

The transiently reduced food consumption observed predominantly during the first treatment days at the high dose level was reversible and therefore regarded to be not adverse.

Necropsy revealed macroscopic hepatic changes in a few intermediate and high dose dams (300 and 1000 mg Test item/kg b.w./day) which were obviously due to increased work load of the liver and considered asnottest item-related.

The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 1000 mg Test item/kg b.w./day. No test item-related effects were noted on the fetus.

However, at 1000 mg Test item/kg b.w./day, increased fetal incidences were noted for variations of the ribs (accessory) and the sternum (bipartite) during skeletal examination according to DAWSON.These findings are classified to be not adverse and could be explained as secondary effects due to the maternal stress caused by materno-toxic effects, e.g. reduced food consumption. Such an effect on skeletal variations is well-known.

No test item-related influence was noted on the reproductive parameters (number of implantation sites, number of resorptions and number of fetuses). No test item-related increase was noted for the number of dead fetuses.

No malformations were noted in the test item-treated groups. No test item-related increases in the fetal incidence of variations were noted during the macroscopic external or internal inspections at laparotomy, or soft tissue examination of the fetal head (according to WILSON). Skeletal examination according to DAWSON revealed no test item-related increase of skeletal retardations.

Justification for classification or non-classification

According to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 and based on the results of the studies Dodecane-12-lactam is not classified.