Oleonitrile

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

8-Feb-2006 to 5-Apr-2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 422 and in compliance with GLP. Read-across from structural identical substance with an aliphatic chain length of only C12.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dodecanenitrile

EC Number:

219-440-1

EC Name:

Dodecanenitrile

Cas Number:

2437-25-4

Molecular formula:

C12H23N

IUPAC Name:

lauronitrile

Details on test material:

Certificate included in report:
Sigma-Aldrich
Product Name Dodecanenitrile, 99%
Product Number U1605
Product Brand Aldrich
CAS Number 2437-25-4
Molecular Formula CH3(CH2)10CN
Molecular Weight 181.32
TEST LOT S14287 RESULTS
QC Acceptance date 22-NOV-2002
APPEARANCE - Clarity CLEAR
APPMRANCE - Colour COLOURLESS
APPEARANCE -State LIQUlD
FT-IR SPECTROSCOPY - FTIR SPECTRUM: CONFORMS TO REFERENCE
FT-IR SPECTROSCOPY - REFERENCE 2(1), 1425C
REFRACTIVE INDEX 1.4363
GAS CHROMATOGRAPHY - PURITY 99.40%

Expiry date 22-AUG-2006
Storage At room temperature (20 ± 5°C)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

NUMBER OF ANIMALS:
40 males, 10 per group
40 females, 10 per group

TEST ANIMALS
- Species: Rat HanRcc:WIST (SPF)
- Source: RCC Ltd, Switzerland
- Age at delivery: 10 weeks
- Age at study initiation: + 7 days acclimatisation
- Weight at study initiation: (P) Males: 292 - 339 g; Females: 174 - 213 g
- Housing: Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz/
Switzerland).
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8-Feb-2006 (delivery) 15-Feb-2006 (initiation) To: 05-Apr-2006 (last necropsies)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 2% Methylcellulose

Details on exposure:

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 10 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

PREPARATION OF DOSING SOLUTIONS:
Frequency of dose formulation At least weekly (7-day stability determined in RCC Study No. A32613)
Storage conditions At room temperature (20 ± 5°C), away from direct sunlight

VEHICLE
Identity: 2% Methylcellulose
Supplier / Batch number: Fluka / No. 449579/1 10105185
Expiry date: JUL-08
Storage conditions: At room temperature (20 ± 5°C), away from direct sunlight
- Amount of vehicle (if gavage): 10 mL/kgBW

ANALYSIS
Samples for determination of concentration, homogeneity and stability (7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of the administration period.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation;
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear; referred to as day 0 post coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility: yes
- Further matings after two unsuccessful attempts: no, female sacrificed
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Content, homogeneity and stability in application formulations.
Purity: Results all within the accepted range of ±20% of the nominal content: Range 88.5% to 109.7%.
Homogeneity: single results deviate not more than 13.3% (<15%) from the corresponding mean.
Stability: For 7 days under storage conditions; recoveries meet the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

Pre-pairing: 14-days
Pairing: Until mating
Gestation: about 21 days
Parturition/lactation: until day 4 post partum
Termination: males: minimum 28 days; pups: day 4 post partum; dams: day 5 post partum.

Frequency of treatment:

Daily

Details on study schedule:

Acclimatization 7 days (minimum) (M/F)
Treatment beginning Day 1 of pre-pairing (M/F)
Pre-pairing 14 days (M/F)
Pairing Until mating (M/F)
Gestation Treatment continued (M); About 21 days (F)
Parturition Treatment continued (M); Expected on day 21 or 22 post coitum (F)
Lactation Treatment continued (M); Until day 4 post partum (F)
Treatment ending One day prior to the actual day of necropsy (at least 28 days of treatment) (M)
On day 4 post partum (F)
Termination After a minimum of 28 days treatment (M)
pups on day 4 post partum; dams on day 5 post partum (F)

During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females.
Females were removed and housed individually if:
a) a copulation plug was observed, and / or
b) the daily vaginal smear was sperm-positive.
This day was designated day 0 post coitum.

If a female was not mated during the 14-day pairing period, this female was paired with a male of the same group which mated already successfully with another female. If mating was not recorded during this additional pairing period (maximum 14 days) the female was sacrificed.

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
0-50-250-1000 mg/kg/day

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on range finding study with 0-250-500-1000 mg/kg/day with 3/sex/dose with Caesarian sections at end gestations. No reproductive effects weer noted in any of the groups. (See attached document)
- Rationale for animal assignment (if not random): random

Allocation: Group:
1 1 – 10 (M) 41 - 50 (F) 0 mg/kg/day (vehicle control)
2 11 - 20 (M) 51 - 60 (F) 50 mg/kg/day
3 21 - 30 (M) 61 - 70 (F) 250 mg/kg/day
4 31 - 40 (M) 71 - 80 (F) 1000 mg/kg/day

Positive control:

No

Examinations

Parental animals: Observations and examinations:

OBSERVATIONS
Mortality rate, Signs and/or symptoms (2/day), Detailed clinical observations (weekly),
Functional observation battery (males shortly before sacrifice, females day 3 or 4 pp) following daily dosing:
a) Cage side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer's reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements/Counts: hindlimb/forelimb grip strength, landing foot splay, rectal remperature.
f) Locomotor activity was measured quantitatively for the same animals. Activity was measured with Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (basing on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes are reported.

DATA RECORDING
Body weight, Food consumption, Lactation and litter data

CLINICAL LABORATORY INVESTIGATIONS
HAEMATOLOGY:
Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet count, Total leukocyte count, Differential leukocyte count, Coagulation: Thromboplastin time & Activated partial thromboplastin time.

CLINICAL BIOCHEMISTRY
Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Aspartate aminotransferase, Alanine aminotransferase, Bile acids, Alkaline phosphatise, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein total, Albumin, Globulin, Albumin/Globulin ratio.

Oestrous cyclicity (parental animals):

No full evaluation estrous cyclicity. Phase of the estrous cycle was determined at vaginal histological examination.

Sperm parameters (parental animals):

No full sperm parametrs. A detailed qualitative examination of the testes was made on extra sections stained with PAS/Hematoxylin, taking into account the tubular stages of spermatogenesis and histopathology of interstitial cell structure.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring :
number and sex of pups, stillbirths, live births, postnatal mortality (up to day 4), presence of gross anomalies, weight gain.

Postmortem examinations (parental animals):

SACRIFICE
Males and females were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital (Eutha 77).
- Male animals: All surviving animals day 29
- Maternal animals: All surviving animals day 5 post partum

GROSS NECROPSY
The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
• liver • brain • adrenals* • heart • thymus • testes (paired) • kidneys (paired) • epididymides (paired) • spleen

HISTOPATHOLOGY
Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Examinations were extended to the animals of the other dosage groups, since treatment-related changes were seen in the highest dose group.
All gross lesions were examined.
Histological examination of ovaries was carried out on any females that did not give birth.

• gross lesions • prostate • testes (in Bouin's fixative) • seminal vesicles with coagulation gland • epididymides (in Bouin's fixative) • ovaries • brain • uterus • spinal cord • thymus • small and large intestines (incl. Peyer's patches) • trachea and lungs (preserved by inflation with fixative and then immersion) • stomach • thyroid • liver • lymph nodes (mesenteric and mandibular) • kidneys • urinary bladder • adrenals • peripheral nerve • spleen • bone marrow • heart

Postmortem examinations (offspring):

Pups were killed by an intraperitoneal injection of sodium pentobarbital.
Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.

Statistics:

Statistical methods applied:
• Means and standard deviations of various data were calculated and included in the report.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

Number of females paired
Number of females mated
Number of non pregnant females
Number of females, which first mating was not detected
Number of females, which died during or after giving birth
Number of females, which were killed for ethical reasons
Number of females, which lost their litters
Number of females which reared their pups until day 4 post partum
Duration of gestion
Implantations
Post-implantation loss

Offspring viability indices:

Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: No signs noted during premating period. 250 & 1000 mg: m&f pushed their heads through the bedding from d4 of the pairing period. At 1000 mg, six females died during or after giving birth.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg: reduced food intake males and females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg: reduced food intake males and females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Same organs as mentioned for gross pathology (see results below)

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

Phase of the estrous cycle was determined at vaginal histological examination.

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

No full sperm examination. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

See table on Fo Animals breeding for F1 litters

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All males survived until the scheduled necropsy. At 250 and 1000 mg/kg/day, all males pushed their heads through the bedding starting on day 4 of the pairing period and continuing until the end of the treatment. This was considered to be test item-related and may be an indicator of discomfort after administration of the test item. No other clinical signs or signs of discomfort were noted.

At 1000 mg/kg/day, six females died or were killed for ethical reasons during or directly after giving birth or by day 4 post partum. In general, all these females showed significant signs of toxicity including piloerection, hunch back or lateral recumbency after giving birth. One female, for which no mating was noted, was killed for ethical reasons three days after its pairing period after exhibiting piloerection and hunch back; this female was determined to be not pregnant.
Only one female showed no clinical signs after giving birth and reared its pups until day 4 post partum, as scheduled. Two females were not pregnant, which was considered to be incidental.
At 50 and 250 mg/kg/day, all females survived until the scheduled necropsy. At 250 and 1000 mg/kg/day, all females pushed their heads through the bedding after administration of the test item starting on day 4 of the pairing period and continuing until the end of the treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 1000 mg/kg/day, mean food consumption was statistically significantly reduced throughout the prepairing period (-19.2% compared to the control group). During this time a slightly reduced mean body weight gain was noted, resulting in a reduced mean absolute body weight (343 g compared to 364 g in the control group). During the pairing period, body weight gain recovered to normal values. However, slightly reduced absolute body weights persisted until the scheduled necropsy.


At 1000 mg/kg/day, mean food consumption was reduced throughout the prepairing and gestation period (-8.1% and -7.1%, respectively, compared to the control group). During the lactation period mean food consumption of the remaining single female was markedly reduced (-64.9% compared to the control group). Mean body weight and mean body weight gain were not affected by treatment with the test item until the end of the gestation. During the lactation period the single female lost 4% of its body weight by day 2 post partum, but showed signs of recovery by study termination.
The single female at 1000 mg/kg/day, similarly to the males at this dose level, showed a reduced number of rearings and a reduced locomotor activity over 30 minutes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Phase of the estrous cycle was determined at vaginal histological examination.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
See attached table.
In groups 1 and 2, all females were mated within the first pairing period. In groups 3 and 4, one female in each group was not mated within the first pairing period, and a second pairing period was erroneously not added. Furthermore, the first mating of one female in group 4 was not detected.
With these three females excluded, the mean precoital times were 3.4, 4.0, 4.1 and 3.8 days in order of ascending dose level. The median precoital time was 3, 4, 3 and 3 days.
Additionally one female in group 2 and two females in group 4 were not pregnant. The resulting fertility indices were 100% in group 1, 90% in groups 2 and 3, and 70% in group 4. Since for male No. 35, which was mated with the non pregnant female No. 75, histopathological findings were noted in both testes, a test item-related effect in group 4 cannot be excluded.

The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.8, 21.6, 21.6 and 22.0 days, in order of ascending dose level.

The mean number of implantations per dam was unaffected by treatment with the test item. The mean numbers of implantations per litter for all dams giving birth were 15.7, 15.1, 15.9 and 15.1, in order of ascending dose level.
In group 4, a test item-related increase in post-implantation loss was noted. The total incidence of post-implantation loss was 53 (compared with 35 in the vehicle control).
Post implantation loss in groups 2 and 3 was statistically significantly lower compared with the control group (5 and 17, respectively), since post implantation loss in the control group was unusually high. Therefore these differences were considered to be incidental.

As a consequence of the increased incidence of post-implantation loss, the mean litter size noted at first litter check in group 4 was reduced (11.6 pups per litter compared with 13.3 pups in the control group, combined data for live and dead pups). In addition to the smaller litter size, the incidence of living pups at first litter check was decreased in this group (7.6 living pups per litter compared with 12.2 in the control group), and the incidence of dead pups at first litter check was increased (4.0 dead pups per litter compared with 1.1 in the control group). Female Nos. 71, 74, and 79 had only dead pups at first litter check and for female Nos. 71 and 74 severely cannibalized pups were additionally found, for which no sex determination was possible. Most probably, the reason for the high incidence of post implantation loss was the bad conditions of the females after parturition, resulting in spontaneous death or the killing for ethical reasons of the females.
For one control group female (No. 46) total litter loss was noted. Although the cause of this litter loss could not be determined, it was considered to be incidental.
Litter sizes and incidence of dead pups at first litter check in groups 2 and 3 were not affected by treatment with the test item.
The gestation indices were 90% in the control group, 100% in groups 2 and 3, and 57.1% in group 4.

In group 4 (100 mg), total litter loss was noted for female Nos. 73, 76 and 77 between days 0 - 4 post partum. The pups of female Nos. 73 and 77 were killed for ethical reasons because female No. 73 was found dead on day 1 post partum and female No. 77 was killed for ethical reasons on day 4 post partum. All pups of female No. 76 died on day 2 post partum together with the dam.

Only when calculated for all dams giving birth the incidence of postnatal loss was statistically significantly increased in group 4. This increase was considered to be attributable to treatment with the test item. When calculated for all dams with live pups on day 4 post partum the incidence of postnatal loss of the remaining single female in group 4 was not increased.
Postnatal loss in groups 2 and 3 was not affected by treatment with the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In group 4(1000 mg), 60% of the males had an enlarged liver and of these males 30% also had a thymus reduced in size. These findings were considered to be test item-related.
In groups 2 and 3, incidental findings such as kidneys with pelvic dilation, a distended urinary bladder and an enlarged liver (one male in group 3) were noted.
Furthermore, black-brown contents in the stomach were noted for 2, 1, 2 and 1 males in order of ascending dose level.

Females at 1000 mg: enlarged liver, stomach with discolourations, crateriform retractions and foci, and adrenal glands enlarged. Secondary to the spontaneous death the start of autolysis, ileum distended with gas, discoloration and incompletely collapsed of the lungs, urinary bladder distended and discolouration of the liver were observed.
The following incidental findings were noted in one or more groups: lymph nodes enlarged, pelvic dilation of the kidney, adrenal glands discoloured, spleen enlarged, duodenum, jejunum, or pancreas thickened and thymus reduced in size or with foci.
Furthermore, black-brown contents were noted in the stomach, jejunum, ileum and/or caecum in 1, 2, 3 and 2 females in order of ascending dose level. Although the cause of this finding could not be determined (there were no intubation errors, the colour of the test item was colourless and 2% Methylcellulose is a common vehicle) it has to be considered incidental.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males 1000 mg: absolute and relative liver weights were statistically significantly increased, which was considered to be test item-related. The statistically significantly higher relative brain and kidney values (organ/body weight ratios) were considered to be a consequence of the reduced body weight.
Absolute and mean organ weights in groups 2 and 3 gave no indication of a test item-related effect.

Females: Since group 4 (1000 mg) consisted only of a single female, no assessment of the organ weights were possible.
In group 2 (250 mg), higher relative kidney values were noted, which were considered to be incidental, due to the absence of a dose-dependency.
Absolute and mean organ weights in group 3 (50 mg) gave no indication of a test item-related effect.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males
Liver: Minimal to moderate, centrilobular to diffuse hepatocellular hypertrophy in group 4.
Thymus: Increased incidence and severity of atrophy/involution in group 4.
Stomach: Increased incidence of ulceration, erosion and mucosal necrosis in the forestomach and glandular stomach in groups 3 and 4. Further lesions of different degrees were: submucosal edema, mucosal edema, squamous hyperplasia, hyperkeratosis, increased submucosal inflammatory infiltrates and inflammatory cell foci in the serosa in groups 3 and 4.
Kidney: Slight increased incidence of tubular basophilia in group 4.
Testes: In one male of group 4 (no. 35), moderate multifocal vacuolar tubular degeneration was recorded in both testes along with minimal cellular detritus in the epididymides. Minimal degrees of inflammatory cell infiltrates, cellular detritus and tubular degeneration were also observed in the prostate gland and there were minimal inflammatory cell infiltrates in both seminal vesicles and coagulating glands. A test item-relation can not be excluded.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Females:
Liver: Minimal to moderate, centrilobular to diffuse hepatocellular hypertrophy in group 4. Moderate centrilobular necrosis in one female of group 4. Slight to moderate hepatocellular apoptosis in group 4.
Spleen, mesenteric and mandibular lymph nodes: Minimal to moderate lymphoid atrophy in group 4.
Thymus: Increased incidence and severity of atrophy/involution in group 4.
Lung: Minimal to moderate lymphoid atrophy of the bronchus associated lymphoid tissue (BALT) in group 4. Moderate multifocal interstitial inflammation in one female of group 4.
Stomach: Increased incidence of ulceration, erosion and mucosal necrosis in the forestomach and glandular stomach in groups 3 and 4. Further lesions of different degrees were: submucosal edema, mucosal edema, squamous hyperplasia, hyperkeratosis, increased submucosal inflammatory infiltrates and inflammatory cell foci in the serosa in groups 3 and 4.
Duodenum: Slight ulceration in one female of group 4.
Kidney: Increased severity of tubular basophilia in group 4.
Adrenal glands: Minimal to moderate hypertrophy of the Zona fasciculata in groups 3 and 4.

Of the above mentioned test item-related findings the following were considered to be of
adverse character: Centrilobular necrosis and apoptosis in the liver in group 4. Ulceration, erosion and mucosal necrosis in forestomach and glandular stomach and duodenum in groups 3 and 4.
Additionally, a variety of other changes were found in this study. They commonly occur in laboratory rats of this strain and age, neither their incidences nor their distribution or morphologic characteristics gave any indication of a treatment-related association.

CLINICAL LABORATORY INVESTIGATIONS
CLINICAL BIOCHEMISTRY
Males 1000 mg: Glucose concentration markedly decreased (-24%), and Sodium concentration slightly increased (+1%)

No biological and/or toxicological relevance is considered to be associated with the marginal to slight changes observed in urea in group 2 and total bilirubin in group 3 since these changes did not show a clear dose relationship.

For females, no assessment of the clinical biochemistry data was possible in group 4, since only one pregnant female survived.
For groups 2 and 3, the assessment of the clinical biochemistry data did not reveal any test item-related effects.

HEMATOLOGY
Males: Hb, Hb distribution width & MCH slightly decreased; Platelet count increased at 250 and 1000 mg (+15% and +23%, respectively)

For females, no assessment of the hematology data was possible in group 4, since only one pregnant female survived.
For groups 2 and 3, the assessment of the hematology data did not reveal any test item-related effects.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At next higher level (250 mg/kg) clinical signs, histopathological findings stomach & adrenal glands.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg: Abnormal findings in litters related to neglect and canibalism.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

1000 mg: Increased postnatal loss for for all dams giving birth.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg: mean pup weight development was impaired during the first 4 days post partum.

Sexual maturation:

not examined

Description (incidence and severity):

Scheduled necropsy on day 4 pp.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Only effect recorded: no milk in the stomach was found in several pups from all groups.

Histopathological findings:

not examined

Details on results (F1)

ABNORMAL FINDINGS AT BIRTH (FIRST LITTER CHECK) AND DURING LACTATION TO WEANING
1000 mg: At first litter check, 2 and 3 pups of female Nos. 73 and 76, respectively, had wounds on different regions of their bodies; for one pup the right paw was already absent (litter 73). These wounds were considered to be the beginning of cannibalism. All pups from female No. 73 were killed for ethical reasons on day 1 post partum and all pups from female No. 76 died on day 2 post partum since both dams died on day 1 and 2 post partum, respectively. During the macroscopical examination most of these pups had no milk in the stomach. This neglect of the pups by their dams was considered to be a consequence of the bad condition in which the dams had been. All pups from litter 77 had no milk in the stomach and were cold to the touch on day 4 post partum. They were killed for ethical reasons after the dam had to be killed for ethical reasons.
For one control group pup, a blue area on the head was noted and in group 3 (250 mg), one pup had a black tip on the tail. These findings were considered to be incidental.

SEX RATIOS
Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item.
The proportion of males at first litter check was 51, 50, 49 and 38% in order of ascending dose level. The proportion of males on day 4 post partum was 50, 50, 50 and 42% in order of ascending dose level (values obtained from table 'All dams with live pups on day 4 post partum). Since there was only a single female in group 4 (1000 mg), these lower values were considered to be incidental.

PUP WEIGHTS TO DAY 4 POST PARTUM
On day 1 post partum, mean pup weights were slightly lower in groups 2, 3 and 4 when compared to the control group. Mean pup weights were 5.7 g in groups 2-4, respectively, and 6.0 g in the control group (combined data for male and female pups). Since the mean number of pups per litter at first litter check was lower in the control group than in groups 2 and 3 (13.3 compared to 14.6 and 14.2, respectively) and the pup weight development was similar (+38.3% compared to +40.4% and 42.1%, respectively) this finding was considered to be incidental.
In group 4 (1000 mg), mean pup weight development was impaired during the first 4 days post partum (+31.6%) resulting in reduced weights on day 4 post partum (7.5 g compared to 8.3 g in the control group). Therefore the reduction in mean pup weight on day 1 and 4 post partum was considered to be test item-related.

PUPS - NECROPSY FINDINGS
During necropsy, no milk in the stomach was found in several pups from all groups. All these pups were dead at first litter check. In group 4, no milk in the stomach was also found in pups which were killed for ethical reasons or died spontaneously.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 250 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The overall NOAEL is 50 mg/kg based on effects in clinical signs, histopathological findings stomach & adrenal glands observed at next higher dose level of 250 mg/kg.
The NOAEL for reproductive effects is 250 mg/kg, based on death and poor health of the females at 1000 mg/kg

Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavageadministration of the test item Dodecanenitrile to rats up to 28 days. Dodecanenitrile wasadministered in a 2% Methylcellulose vehicle at dosages of 50, 250, and 1000 mg/kg bodyweight/day, and controls received the vehicle only. Dose volume was 10 mL/kg. Dodecanenitrilewas administered to male rats for at least 28 days and to female rats for 14 days prior to pairing,through the prepairing, pairing and gestation periods until the F1 generation reached day 4 postpartum.

 

At 1000 mg/kg/day, six females died or were killed for ethical reasons during or directly aftergiving birth or by day 4 post partum. In general, all these females showed significant signs oftoxicity including piloerection, hunch back or lateral recumbency after giving birth. One female,for which no mating was noted, was killed for ethical reasons three days after its pairing periodafter exhibiting piloerection and hunch back. Other treatment related effects at 1000 mg/kg/daywere pushing their heads through the bedding after administration, reduced mean foodconsumption and reduced mean body weight (males only). Secondary to the parental toxicity,reduced open field activity and reduced locomotor activity, reduced body temperature (onlymales), increased incidence of post implantation loss, reduced mean litter size, increasedincidence of dead pups at first litter check and increased incidence of postnatal loss wereobserved. Macroscopically, for males an enlarged liver and a thymus reduced in size werenoted, whereas for females, stomachs with discolourations, crateriform retractions and foci, andenlarged lymph nodes and adrenal glands were noted. Grossly, mean absolute and relative liverweights were increased. For the females no assessment of the organ weights was possible,since only one female survived. Histopathological findings were observed in liver, spleen,thymus, stomach, duodenum, kidney and adrenal glands in both sexes, but mainly in females.Clinical biochemistry data revealed several effects for glucose, sodium, hemoglobin and mean corpuscular hemoglobin concentration, for hemoglobin concentration distribution width and forplatelet count in males. No assessment of the clinical biochemistry data and hematology datawere possible for females. Mean pup weight on day 0 post partum and mean pup weightdevelopment were reduced at 1000 mg/kg/day.

 

At 250 mg/kg/day, all animals pushed their heads through the bedding after administration andopen field activity and locomotor activity were reduced. Histopathological findings wereobserved in the stomach and adrenal glands. Clinical biochemistry data revealed several effects for hemoglobin and corpuscular hemoglobin concentration and platelet count in males.

 

Based on these data, a NOEL (no observed effect level) can be established at 50 mg/kg/day.