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Description of key information

Based on the results of a OECD 422 study: NOAEL(systemic) > 250 mg/kg bw/day; NOAEL(local): > 75 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: animals of comparable size and weight
- Housing: 5 animals/sex and cage pre-mating, one male and one female during mating, females individually after mating, males group-housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was chosen based on trial formulations performed at WIL Research Europe
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day dose range finding study.
- Rationale for animal assignment (if not random): by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS (for mortality) Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made in all animals, at least immediately after dosing (on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were conducted at least immediately after dosing.

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

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY:
Blood samples were collected from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
Furthermore, from the selected 5 animals/sex/group an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Since histopathological examination of the thyroid glands did not reveal any treatment-related changes, all samples were discarded at finalization of the study report without further investigation.
Parameters examined: White blood cells, Differential leucocyte count, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY:
Blood samples were collected from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
Furthermore, from the selected 5 animals/sex/group an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Since histopathological examination of the thyroid glands did not reveal any treatment-related changes, all samples were discarded at finalization of the study report without further investigation.
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the selected 5 animals/sex/group:
- hearing ability
- pupillary reflex
- static righting reflex
- grip strength
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported.
During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water).

The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable), i.e. as soon as possible after dosing, and before blood sampling.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs will be collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and female mammary gland area), Spleen, Femur including joint Sternum with bone marrow, Heart, Stomach, Ileum, Testes, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination [Examination of the mammary gland area was required for females nos. 71 and 76 with total litter loss.].

HISTOPATHOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

A peer review on the histopathology data was performed by a second pathologist.

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously.
- All gross lesions of all animals (all dose groups).
- Stomach the selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in Group 4.
- Thymus of the selected 5 animals of Groups 2 and 3 (females), based on (possible) treatment-related changes in this organ in Group 4.
- The reproductive organs of all animals of Groups 1 and 4, and of males 31 and 36 (Group 4) and females 71 and 763 (Group 4); both females had a total litter loss.
Other examinations:
Organ weights
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis non-parametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among animals of the 25, 75 and 250 mg/kg bw/day dose group was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.

Incidental findings that were noted included regurgitation together with rales at a slight degree, chromodacryorrhoea (snout) and scabbing on different parts of the body. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no mortalities that were related to the test substance.

One female at 75 mg/kg bw/day and one female at 250 mg/kg bw/day were found dead after dosing on Day 9 of the pre-mating period. Before death, the Group 4 female showed severe restlessness together with breathing difficulties (laboured respiration and gasping). No clinical signs were noted for the Group 3 female. Body weights and food consumption were within the normal range. Gross findings at necropsy included perforation of the oesophagus (granulation tissue and an inflammatory process at microscopic examination), discoloration of the lungs (reddish or dark-red) and Harderian glands (pale) and/or (yellowish) watery-cloudy fluid in the thoracic cavity for both females. These findings were suggestive of a gavage accident as cause of death. In this context it is noteworthy that animals treated with the test substance (Groups 2-4) were more restless during dosing than controls.

Two females at 250 mg/kg bw/day had to be euthanized after total litter loss on Day 1 of lactation.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in body weight and body weight gain were noted.

Body weights and body weight gains were statistically significantly lower in males at 75 and 250 mg/kg bw/day on Day 8 of the pre-mating period and thereafter. However, the differences to controls were slight, and values remained within the range considered normal for rats of this age and strain (5-95% confidence interval body weight gain on mating Day 15: 11-30%). Therefore, these differences were considered not to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were noted.

The slightly, but statistically significantly lower food intake (absolute and/or relative to body weight) noted for females at 75 mg/kg bw/day from Days 7-11 post-coitum and for females at 250 mg/kg bw/day from Days 0-4 and 7-11 post-coitum was considered to be of no toxicological relevance, as values remained within the range considered normal for rats of this age and strain and/or changes occurred in the absence of a treatment-related distribution.

At the individual level, both absolute and relative food intake was lower for three females at 250 mg/kg bw/day (nos. 73, 75 and 77) during lactation. This was at least for females 73 and 75 in part due to the relatively small litter size (5 and 4 pups, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.

The higher relative numbers of reticulocytes for males at 75 and 250 mg/kg bw/day, and slightly lower prothrombin time (PT) and activated partial thromboplastin time (APTT) for males at 25 mg/kg bw/day were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

For both males and females, a lower concentration of bilirubin was noted at 250 mg/kg bw/day as compared to controls. Mean values were at the lower end of the historical range of data (1.6 µmol/L for males and 1.7 µmol/L for females in this study versus a historical control (5-95% confidence interval: 1.7-2.60 µmol/L for males and 1.6-3.0 µmol/L for females). However, in the absence of corroborative changes on other parameters, this finding was considered of no toxicological relevance.

Any other statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain. These changes were seen for males only and included slightly lower concentrations of total protein (at 25 mg/kg bw/day) and cholesterol (at 250 mg/kg bw/day) and a slightly higher concentration of potassium (at 75 mg/kg bw/day).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

Locomotor activity as determined by total movements and ambulations was unaffected by treatment up to 250 mg/kg bw/day (both sexes). Mean values remained within the normal range of biological variation. All groups showed a comparable habituation profile with high activity in the first interval that decreased over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A trend towards lower thymus organ weights (absolute and relative to body weight) was noted at 250 mg/kg bw/day (both sexes) as compared to controls. This change was relatively slight and reached statistical significance for the mean value of absolute thymus organ weight in females only.

The lower absolute weight of thyroid for males at 25 and 75 mg/kg bw/day and lower absolute weight of epididymides recorded at 250 mg/kg bw/day were considered not to be toxicologically relevant, since they occurred in the absence of a treatment-related distribution, no corroborative changes at the microscopic level were noted and/or values remained within the normal range of biological variation.

In line with the lower body weights noted for males at 75 and 250 mg/kg bw/day as compared to controls during in-life, also terminal body weights were statistically significantly lower in males of the mid and high dose group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings.

Incidental findings among control and treated animals included nodules (yellowish, soft) on the tail of the epididymides, isolated, tan or dark-red foci on one preputial gland, clitorial glands and on the glandular mucosa of the stomach, tan discoloration of preputial and clitorial gland (unilateral), small size of adrenal gland and liver lobe, scab formation on the skin and alopecia, and/or pelvic dilation of the kidneys. These necropsy findings were considered to be of no toxicological relevance due to their isolated occurrence.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic findings were noted:

Stomach (both sexes)
- An increased incidence and severity of hyperplasia of the squamous epithelium with hyperkeratosis was recorded at 250 mg kg bw/day in 6/6 males (moderate) and in 5/5 terminal females (2: minimal, 2: slight, 1: moderate).
- A lymphogranulocytic inflammation of the forestomach at a minimal degree was recorded at 250 mg/kg bw/day in 6/6 males and 2/5 terminal females.

Thymus (females)
- A slightly increased incidence and severity of lymphoid atrophy was recorded in 3/5 terminal females (1: minimal, 2: slight) at 250 mg/kg bw/day.
Minimal lymphoid atrophy of the thymus was also recorded in a single female of each the control and 75 mg/kg bw/day group and a single male at 250 mg/kg bw/day.

The minimal lymphoid atrophy of the thymus in single animals and all remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.

Examination of the reproductive organs of the animals (incl. assessment of the integrity of the spermatogenetic cycle for males) did not reveal any abnormalities up to 250 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects in the fore stomach
Critical effects observed:
not specified

Repeated administration of alpha,alpha’-Propylenedinitrilodi-o-cresol to male and female Wistar Han rats caused salivation after dosing, which was transient and occurred at a very low to low incidence at 25 and 75 mg/kg bw/day, and at a higher incidence at 250 mg/kg bw/day. This finding was considered to be probably a physiological response and therefore not as adverse.

 

An increased incidence and severity of hyperplasia of the squamous epithelium with hyperkeratosis (up to a moderate degree) and lymphogranulocytic inflammation of the forestomach (minimal degree) were recorded at 250 mg kg bw/day (both sexes). This finding is suggestive for an irritant potential of the test substance. In line with this, macroscopic abnormalities indicative for irritation of the mucosa of the stomach were noted at 300 and 800 mg/kg bw/day (both sexes) in the previous dose range finding study.

 

The trend towards lower thymus organ weights (absolute and relative to body weight) noted at 250 mg/kg bw/day (both sexes) together with a slightly increased incidence and severity of lymphoid atrophy (up to a slight degree; females only) was not considered as an adverse effect, since changes were relatively slight. They were most likely secondary to stress caused by the local effects of the test substance on the stomach.

 

No adverse effects were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, clinical appearance, functional observations (incl. locomotor activity), body weight, food consumption, clinical laboratory investigations (haematology and clinical biochemistry), and macroscopic examination

Conclusions:
NOAEL of at least 250 mg/kg bw/day for systemic toxicity, 75 mg/kg bw/day based on local irritation at the stomach
Executive summary:

Repeated administration of alpha,alpha’-Propylenedinitrilodi-o-cresol to male and female Wistar Han rats caused salivation after dosing, which was transient and occurred at a very low to low incidence at 25 and 75 mg/kg bw/day, and at a higher incidence at 250 mg/kg bw/day. This finding was considered to be probably a physiological response and therefore not as adverse.

 

An increased incidence and severity of hyperplasia of the squamous epithelium with hyperkeratosis (up to a moderate degree) and lymphogranulocytic inflammation of the forestomach (minimal degree) were recorded at 250 mg kg bw/day (both sexes). This finding is suggestive for an irritant potential of the test substance. In line with this, macroscopic abnormalities indicative for irritation of the mucosa of the stomach were noted at 300 and 800 mg/kg bw/day (both sexes) in the previous dose range finding study.

 

The trend towards lower thymus organ weights (absolute and relative to body weight) noted at 250 mg/kg bw/day (both sexes) together with a slightly increased incidence and severity of lymphoid atrophy (up to a slight degree; females only) was not considered as an adverse effect, since changes were relatively slight. They were most likely secondary to stress caused by the local effects of the test substance on the stomach.

 

No adverse effects were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, clinical appearance, functional observations (incl. locomotor activity), body weight, food consumption, clinical laboratory investigations (haematology and clinical biochemistry), and macroscopic examination.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of alpha,alpha’-Propylenedinitrilodi-o-cresol was conducted in rats by oral gavage according to OECD 422 guideline and GLP (WIL Research Europe, 2013).

Based on the results of a 14-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 25, 75, or 250 mg/kg bw/day.

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 25, 75, or 250 mg/kg bw/day. Rats of the control group received the vehicle, Polyethylene glycol 400, alone. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)), body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results

Repeated administration of alpha,alpha’-Propylenedinitrilodi-o-cresol to male and female Wistar Han rats caused salivation after dosing, which was transient and occurred at a very low to low incidence at 25 and 75 mg/kg bw/day, and at a higher incidence at 250 mg/kg bw/day. This finding was considered to be probablya physiological response and therefore not as adverse.

 

An increased incidence and severity of hyperplasia of the squamous epithelium with hyperkeratosis (up to a moderate degree) and lymphogranulocytic inflammation of the forestomach (minimal degree) were recorded at 250 mg kg bw/day (both sexes). This finding is suggestive for an irritant potential of the test substance.

The trend towards lower thymus organ weights (absolute and relative to body weight) noted at 250 mg/kg bw/day (both sexes) together with a slightly increased incidence and severity of lymphoid atrophy (up to a slight degree; females only) was not considered as an adverse effect, since changes were relatively slight. They were most likely secondary to stress caused by the local effects of the test substance on the stomach.

 

No adverse effects were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, clinical appearance, functional observations (incl. locomotor activity), body weight, food consumption, clinical laboratory investigations (haematology and clinical biochemistry), and macroscopic examination).

 

Reproductive results

Two females at 250 mg/kg bw/day had a total litter loss on Day 1 of lactation, resulting in a gestation index of only 77.8% for Group 4 as compared to 100% for the remaining groups. The reason for the total litter loss could not be established as part of this study. Based on the in-life data there were no indications for a poor condition of these two females, and examination of thereproductive organs of the animals that failed to deliver healthy offspring (male 31, 36, female 71, 76) did not reveal any abnormalities.

 

No treatment-related toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

 

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

 

Developmental results

Total litter loss was noted for two dams at 250 mg/kg bw/day at first litter check, leading to a decreased gestation index of 77.8%. In addition, excluding these two dams, mean live litter size was slightly lower in this group as compared to the control group.

 

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e.duration of pregnancy, parturition, maternal care, sex ratio of the pups, and earlypostnatalpup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: at least 250 mg/kg bw/day for systemic toxicity, 75 mg/kg bw/day based on local irritation at the stomach;

Reproduction NOAEL: 75 mg/kg bw/day;

Developmental NOAEL:75 mg/kg bw/day.

Range-Finder:

In a GLP compliant 14-day dose range finding study (WIL Research Europe, 2012) Alpha,alpha’-Propylenedinitrilodi-o-cresol was administered by daily oral gavage to Wistar Han rats (4 animals/sex/group) at dose levels of 300 and 800 mg/kg/day. Animals of the control group received the vehicle, polyethylene glycol 400, alone. Controls and animals at 300 mg/kg bw/day were exposed for 14 days. Treatment at 800 mg/kg bw/day was stopped after a maximum of 4 days, due to unacceptable high toxicity. Formulation analysis showed that the formulations for Groups 1 and 2 were prepared accurately. The Group 2 formulation was homogenous and stable for at least 6 hours at room temperature. At 800 mg/kg bw/day, two females were found dead one each on Days 2 and 3, and two males were found dead on Day 4. Since toxicity at this dose level was too high, the remaining two females and two males were euthanized on Days 3 and 4, respectively. In the high dose group, clinical signs consisted of slight to severe lethargy, severe clonic spasms, muscle twitching, abnormal, flat or hunched posture, slightly uncoordinated movements, gasping, slightly laboured respiration, ptosis, piloerection, chromodacryorrhoea and/or slight salivation. A body weight loss of 5-9% was recorded on treatment Day 3. In addition, a higher water intake was noted for rats at 800 mg/kg bw/day as compared to the 300 mg/kg bw/day treated rats and controls on Days 3 and/or 4 (subjective appraisal). At necropsy, isolated to many reddish or dark-red foci were noted on the stomach of all animals. In addition, one female had enlarged, pale adrenal glands. At the lower dose level of 300 mg/kg bw/day, no toxicological relevant changes were noted with respect to clinical appearance, body weights and food consumption. Clinical laboratory investigations revealed a slightly higher reticulocyte count, and slightly higher values of creatinine and inorganic phosphates (both sexes) and urea (females only). Gross findings at necropsy included an irregular surface of the forestomach for two males and three females. This finding is indicative for the irritant potential of the test item in the forestomach at 300 mg/kg bw/day and was confirmed by the results of additional histopathological examination of the stomachs at the microscopic level. Organ weights (absolute and relative to body weight) for liver and kidneys were slightly higher in females only. In addition, two out of the four females had a slightly higher adrenal gland organ weight (absolute and relative to body weight). Based on these results, dose levels of 0, 25, 75, and 250 mg/kg bw/day were selected for the definitive OECD 422 study, taking into account the longer administration period and inclusion of pregnant animals. To avoid possible local effects, preventative measures (i.e. wiping of the flexible catheter prior to dosing and water flush after dosing) were taken to reduce the irritant effect of the test compound. In the absence of toxicologically relevant clinical signs at 300 mg/kg bw/day, clinical and functional observations were conducted at least directly after dosing in the main study.

Justification for classification or non-classification

Based on the available data, the test substance is not classified with regard to repeated dose toxicity according to Regulation (EC) No 1272/2008 (CLP), respectively.

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