Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Endpoint summary

Description of key information

LOAEL (rat) = 10 mg/kg bw/day

RA from Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4)

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 Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

not specified

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
- 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): approximately 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 bw

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 gestation, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

100 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 results of the dose range finding study where dose levels of 100, 300 and 1000 mg/kg bwday were assessed.

Positive control:

No

Observations and examinations performed and frequency:

PARENTAL ANIMALS
- Mortality / Viability: At least twice daily.

CLINICAL SIGNS: 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.
FOB: 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).

BODY WEIGHT: 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 of gestation 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 of gestation and on Days 1 and 4 of lactation.

HAEMATOLOGY: Yes
- 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.

Sacrifice and pathology:

- Necropsy: 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.
- 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: 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 lymphnodes (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

Description (incidence and severity):

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 at this dose level.

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 weights were moderately increased in both sexes at 100 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged spleen was observed in one male and 4 females at 100 mg/kg bw/day. Enlarged mesenteric lymph nodes were present in 5 males and all females at 100 mg/kg bw/day, in 6 females at 30 mg/kg bw/day and in 2 females at 10 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 bw/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 bw/day (up to a moderate degree) and in females at 30 and 100 mg/kg bw/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 bw/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 bw/day.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

Treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg bw/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 bw/day were considered to be due to these inflammatory findings. Therefore, the LOAEL was set at 10 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Hazard assessment is conducted by means of read-across from a structural analogue/surrogate. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for Read-across

There are no data available regarding repeated dose toxicity for Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5). Therefore, read-across from the appropriate substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfill the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.6. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed analogue justification for the read-across approach is provided in IUCLID Section 13.

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD 422 and under GLP conditions, 10 male and 10 female Wistar rats received Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4) by gavage at dose levels of 10, 30 and 100 mg/kg bw/day; dose selection was based on the results of a preliminary range-finding study (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 gestation, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Parental animals were observed at least twice daily for mortality and daily for evidence of clinical signs. Body weights and food consumption were recorded weekly. At necropsy, haematological and clinical biochemistry parameters were determined. In addition, organ samples were taken from all animals and the organ weights were recorded from 5 animals/sex/dose level at necropsy. Histopathological examination was performed for control and high-dose groups, and for all dose groups when gross lesions were observed.

Mortality and clinical signs were not observed up to the end of the study period. Slightly increased leucocyte counts in males and females at 100 mg/kg bw/day, primarily due to increased neutrophil counts, were observed. Increased leucocyte counts were also reported for females at 30 mg/kg bw/day, whereas the percentage of neutrophils was increased in both sexes at this dose level. Spleen weights were moderately increased in both sexes at 100 mg/kg bw/day. In correlation, enlarged spleen was observed in one male and 4 females at 100 mg/kg bw/day. Enlarged mesenteric lymph nodes were present in 5 males and all females at 100 mg/kg bw/day, in 6 females at 30 mg/kg bw/day and in 2 females at 10 mg/kg bw/day. 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 cortex (inflammatory lymphocytic cells) were noted in males and females, respectively, and were considered to be treatment-related. Since these effects were observed in both sexes of all dose groups, the LOAEL was set at the lowest dose level of 10 mg/kg bw/day.

Justification for classification or non-classification

The available data from the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD 422 conducted with the analogue substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4) 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 under Regulation (EC) No.1272/2008 will be made.