Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Endpoint summary

Description of key information

In a GLP compliant study according to OECD422, treatment with Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed parental toxicity all dose levels. Therefore, a parental No Observed Adverse Effect Level (NOAEL) could not be established.

Key value for chemical safety assessment

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:

26 July 2012 - 24 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (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, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males: XXg, Females: XXg
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

- Amount of vehicle: 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
In the Group 1 formulation, no test substance was detected. The concentrations analysed in the formulations of Group 2, 3 and Group 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2, 3 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of Group 2, 3 and Group 4 formulations after storage yielded an absolute relative difference ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily for 7 days per week

Remarks:

Doses / Concentrations:
10, 30, 100 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of the dose range finding study where dose levels of 0, 100, 300 and 1000 mg/kg b.w./day were assessed.
- Post-exposure recovery period in satellite groups: no

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, immediately after dosing (0-15 minutes 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 postcoitum and on Days 1 and 4 of lactation.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters were examined: White blood cells, Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets. The following clotting parameters were determined in plasma: Prothrombin Time, Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: all
The following clinical biochemistry parameters were determined: 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
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period.
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: 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).

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. Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7.
Female no. 57 which failed to deliver: Post-coitum Day 27 (female with evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
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.

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate (weighed when fixed for at least 24 hours), Seminal vesicles including coagulating glands (weighed when fixed for at least 24 hours), Thyroid including parathyroid (weighed when fixed for at least 24 hours). All remaining males: Epididymides, Testes.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, (Aorta), Brain - cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), (Male and Female mammary gland area), Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions. The epididymides, eyes and testes were fixed in modified Davidson's solution and Milli-Ro water and transferred to formalin after fixation for at least 24 hours. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

The following slides were examined by a pathologist:
(1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
(2) The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis.
(3) All gross lesions of all animals (all dose groups).
(4) Mesenteric lymph nodes (males and females) and adrenal glands (females) of all selected 5 animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
(5) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) from all animals of Groups 1 and 4 and from male no.17 (Group 2) that failed to sire and female no. 57 (Group 2) that failed to deliver healthy pups.
(6) The preserved organs and tissues of the animals of all dose groups which died spontaneously. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Other examinations:

PUPS:
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
- Necropsy pups: Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

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.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences
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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

spleen

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

spleen and mesenteric lymph nodes

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

spleen and adrenal glands

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. No clinical signs of toxicity were noted during the observation period. Incidental cases of salivation, alopecia and piloerection 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.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

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

HAEMATOLOGY
Higher white blood cell counts were recorded for males at 100 mg/kg and for females at 30 and 100 mg/kg b.w. day (not statistically significant for females). Relative and absolute neutrophil counts were increased in males and females at 30 and 100 mg/kg b.w./day (not statistically significant for females at 30 mg/kg b.w./day).
The statistically significantly lower relative lymphocyte counts in males and females at 30 and 100 mg/kg b.w./day were ascribed to the higher relative neutrophil counts at these dose levels. The lower red blood cell counts and lower haemoglobin levels in females at 30 mg/kg occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes were therefore considered to be of no toxicological relevance. The same applies to the higher absolute monocyte and eosinophil counts in males at 100 mg/kg b.w./day and lower absolute monocyte counts in females at 10 mg/kg b.w./day.

CLINICAL CHEMISTRY
No toxicologically relevant changes in clinical biochemistry parameters were noted.
The higher aspartate aminotransferase activity in males, higher alanine aminotransferase activity in females, lower albumin level in males, higher inorganic phosphate level in males and higher potassium level (not statistically significant) in females (all occurring at 100 mg/kg b.w./day) were slight
in nature and remained essentially within the range considered normal for rats of this age and strain. These differences were therefore not considered to be toxicologically relevant. Alanine aminotransferase activity in males at 100 mg/kg b.w./day also was higher compared to control values, but the individual variation was high and the mean remained with the normal range. The statistically significant lower calcium level in males at 10 and 30 mg/kg b.w./day occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. No toxicological relevance was therefore ascribed to these changes.

NEUROBEHAVIOUR
Hearing ability, static righting reflex and grip strength were normal in all selected animals. No toxicologically relevant effects on pupillary reflex were noted. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in
the first interval that decreased over the duration of the test period. One male at 30 mg/kg (no.24) showed absence of myosis. Given the incidental and unilateral nature of this finding, this was considered to be of no toxicological relevance.

ORGAN WEIGHTS
Spleen weights (absolute and relative) were higher in males and females at 100 mg/kg b.w./day (relative mean spleen weight was approximately 26 and 40% higher than controls in males and females, respectively).
The statistically significant higher absolute and relative seminal vesicle weight at 10 mg/kg b.w. day, the higher relative kidney weights in males at 10 and 100 mg/kg b.w./day, occurred in the absence of a dose-related trend. The statistically significant higher relative epididymides weight at 100 mg/kg
b.w./day remained well within the range considered normal for rats of this age and strain (as well as absolute epididymides weight at this dose), and no histopathological correlates were noted. No toxicological relevance was therefore ascribed to these changes.

GROSS PATHOLOGY
Enlarged mesenteric lymph nodes were noted in 5/10 males and all females at 100 mg/kg b.w./day, in 6/10 females at 30 mg/kg b.w./day and in 2/10 females at 10 mg/kg b.w./day. Enlargement of the spleen was noted in 1/10 males and 4/10 females at 100 mg/kg b.w./day. The incidence of other macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend and/or occurred in the absence of histopathological correlates. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related microscopic findings were noted:
Mesenteric lymph nodes:
- Foamy macrophages with/without fibrosis was recorded at 10 mg/kg b.w./day in 5/5 males (2 minimal, 3 slight) and 6/7 females (4 slight, 2 moderate), at 30 mg/kg b.w./day in 5/5 males (3 moderate, 2 marked) and 8/8 females (1 slight, 2 moderate, 5 marked) and at 100 mg/kg b.w./day in 5/5 males (1 moderate, 4 marked) and in 10/10 females (2 moderate, 8 marked).
- Sinus ectasia was recorded at 10 mg/kg in 1/7 females (moderate), at 30 mg/kg b.w./day in 1/8 females (slight),and at 100 mg/kg b.w./day in 5/10 females (2/10 minimal and 3/10 slight). This finding was considered secondary to the occurrence of foamy macrophages.
- Focal necrosis, within the aggregates of foamy macrophages with/without fibrosis was recorded at 10 mg/kg b.w./day in 1/5 males (minimal), at 30 mg/kg b.w./day in 4/5 males (1 minimal, 2 slight, 1 moderate) and 8/8 females (1 minimal, 3 slight, 4 moderate) and at 100 mg/kg b.w./day in 4/5 males (1 minimal, 3 slight) and in 10/10 females (1 slight, 8 moderate, 1 marked).
- Macrophage foci were recorded at 10 mg/kg b.w./day in 4/5 males (2 slight, 1 moderate, 1 marked) and 6/7 females (2 minimal, 4 slight), at 30 mg/kg b.w./day in 5/5 males (1 minimal, 3 slight, 1 marked) and 5/8 females (all slight) and at 100 mg/kg b.w./day in 4/5 males (1 minimal, 2 slight, 1 moderate) and in 4/10 females (1 slight, 3 moderate).
Adrenals:
- Inflammatory lymphocytic cells in the adrenal cortex were recorded at 10 mg/kg b.w./day in 1/5 females (minimal), at 30 mg/kg b.w./day in 2/5 females (1 minimal, 1 slight) and in 100 mg/kg b.w./day in 4/5 females (3 minimal, 1 slight).
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.

Dose descriptor:

NOAEL

Effect level:

other: not identified

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed parental toxicity at all dose levels. Therefore, a parental No Observed Adverse Effect Level (NOAEL) could not be established.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

For the reproduction/developmental data obtained in this study see chapter 7.8

Conclusions:

Treatment with the test item by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed parental toxicity at all dose levels. These findings were confined to histopathological lesions in the mesenteric lymph nodes (foamy macrophages with/without fibrosis, sinus ectasia, focal necrosis within the aggregates of foamy macrophages with/without fibrosis and macrophage foci) and adrenal gland cortex (inflammatory lymphocytic cells). The higher neutrophil counts (and white blood cell counts) at 30 and 100 mg/kg b.w./day were considered to be due to these inflammatory
histopathological changes. A No Observed Adverse Effect Level (NOAEL) could not be established. The LOAEL is considered to be 10 mg/kg bw/day.

Executive summary:

In a Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was peformed according to OECD guideline 422 in rats by oral gavage. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 10, 30 and 100 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No clinical signs of intoxication were observed in parental animals. Body weight development and food consumption were not affected by administration of the test compound.

Hematological examinations revealed slightly increased leucocyte counts in males and females at 100 mg/kg b.w./day, primarily due to increased neutrophil counts. Increased leucocyte counts were also present in females at 30 mg/kg b.w./day, and the percentage of neutrophils was increased in both sexes at this dose. No toxicologically relevant changes were observed by clinical chemistry

examinations. Spleen weights were moderately increased in both sexes at 100 mg/kg b.w./day. Enlarged spleen was observed in one male and 4 females at 100 mg/kg b.w./day. Enlarged mesenteric lymph nodes were present in 5 males and all females at 100 mg/kg b.w./day, in 6 females at 30 mg/kg b.w./day and in 2 females at 10 mg/kg b.w./day. Treatment-related microscopic findings were noted in the mesenteric lymph nodes and adrenal gland cortex (all dose groups). Findings in the mesenteric lymph nodes consisted of:

− foamy macrophages with/without fibrosis (up to a marked degree), which was correlated with enlargement of the mesenteric lymph nodes at necropsy.

− Sinus ectasia (up to a moderate degree) 10, 30 and 100 mg/kg b.w./day, which was considered secondary to the occurrence of foamy macrophages.

− Focal necrosis within the aggregates of foamy macrophages with/without fibrosis of males at 10, 30 and 100 mg/kg b.w./day (up to a moderate degree) and in females at 30 and 100 mg/kg b.w./day (up to a marked degree).

− Macrophage foci up to a moderate degree (females) or marked degree (males) at 10, 30 and 100 mg/kg b.w./day.

Findings in the adrenal cortex consisted of inflammatory lymphocytic cells (up to a slight degree) in females at 10, 30 and 100 mg/kg b.w./day.

No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg b.w./day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites). Histopathologically, the assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, and no abnormalities were seen in the reproductive organs of the animal that failed to deliver healthy offspring. No developmental toxicity was observed up to the highest dose level tested (100 mg/kg b.w./day).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

In conclusion, treatment with the test article by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed parental toxicity at all dose levels. These findings were confined to histopathological lesions in the mesenteric lymph nodes (foamy macrophages with/without fibrosis, sinus ectasia, focal necrosis within the aggregates of foamy macrophages with/without fibrosis and macrophage foci) and adrenal gland cortex (inflammatory lymphocytic cells). The higher neutrophil counts (and white blood cell counts) at 30 and 100 mg/kg b.w./day were considered to be due to these inflammatory

histopathological changes. A parental No Observed Adverse Effect Level (NOAEL) could not be established. No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg b.w./day. Therefore, a reproduction and developmental NOAEL of at least 100 mg/kg b.w./day was derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

10 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

In a GLP compliant combined 28-days repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422), the test item was given to rats by oral gavage at concentrations of 10, 30 and 100 mg/kg bw (WIL Research 2013). Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. No clinical signs of intoxication were observed. Body weight development and food consumption were not affected by administration of the test compound. Hematological examinations revealed slightly increased leucocyte counts in males and females at 100 mg/kg bw/day, primarily due to increased neutrophil counts. Increased leucocyte counts were also present in females at 30 mg/kg bw/day, and the percentage of neutrophils was increased in both sexes. No toxicologically relevant changes were observed by clinical chemistry examinations. Spleen weights were moderately increased in both sexes at 100 mg/kg bw/day. Enlarged spleen was observed in one male and 4 females at 100 mg/kg b.w./day. Enlarged mesenteric lymph nodes were present in 5 males and all females at 100 mg/kg b.w./day, in 6 females at 30 mg/kg b.w./day and in 2 females at 10 mg/kg b.w./day. Treatment-related microscopic findings were noted in the mesenteric lymph nodes and adrenal gland cortex (all dose groups). Findings in the mesenteric lymph nodes consisted of: (1) foamy macrophages with/without fibrosis (up to a marked degree), which was correlated with enlargement of the mesenteric lymph nodes at necropsy (seen in most animals at 100 mg/kg b.w./day, most females at 30 mg/kg b.w. day and two females at 10 mg/kg b.w. day). (2) Sinus ectasia (up to a moderate degree) at 10, 30 and 100 mg/kg b.w./day, which was considered secondary to the occurrence of foamy macrophages. (3) Focal necrosis within the aggregates of foamy macrophages with/without fibrosis of males at 10, 30 and 100 mg/kg b.w./day (up to a moderate degree) and in females at 30 and 100 mg/kg b.w./day (up to a marked degree). (4) Macrophage foci up to a moderate degree (females) or marked degree (males) at 10, 30 and 100 mg/kg b.w./day. Findings in the adrenal cortex consisted of inflammatory lymphocytic cells (up to a slight degree) in females at 10, 30 and 100 mg/kg b.w./day. Treatment with the test material by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg body weight/day revealed systemic toxicity at all dose levels. Therefore, a parental No Observed Adverse Effect Level (NOAEL) could not be established.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is not sufficient for the purpose of classification under Directive 67/548/EEC. Therefore, a 90d repeated dose toxicity is proposed. Based on the data of this subchronic study a decision on the classification under Directive 67/548/EEC will be made.

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are insufficient for the purpose of classification under Regulation (EC) No.1272/2008. Therefore, a 90d repeated dose toxicity is proposed. Based on the data of this subchronic study a decision on the classification on the classification under Regulation (EC) No.1272/2008 will be made.