Reaction product aqueous Phosphoric Acid, 2-Butoxyethanol and 4,4'-Isopropylidenediphenol-Epichlorohydrin

Endpoint summary

Description of key information

A reliable GLP repeated dose/reproductive screen conducted in accordance with OECD Test Guideline 422 is available assessing the effects of the test substance on repeated dose endpoints.

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

Type of information:

experimental study

Adequacy of study:

key study

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

HSD

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: Ordered at 216 to 239 g (males) and 193 to 202 g (females)
- Fasting period before study: No
- Housing: From arrival to mating, animalswere housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages
with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. Nesting material was provided inside suitable bedding bags in association to suitable nesting material, as necessary. Nesting material was changed at least 2 times a week.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet was offered ad libitum throughout the study.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 27 days

DETAILS OF FOOD AND WATER QUALITY: Contained no contaminants believed to impact the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Details on route of administration:

Standard as per OECD guideline

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The required amounts of the test substance were suspended in the vehicle. Before dosing each dosing formulation (Groups 2, 3 and 4) were maintained under magnetic stirring at approximately 37°C until a clear suspension was obtained. Thereafter the formulations were maintained under magnetic stirring at room temperature until the last animals were dosed.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle as per OECD guidelines.
- Concentration in vehicle: 25, 62.5 and 125 mg/mL.
- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Assessment of the formulation procedure was checked in the range from 25 to 125mg/mL (concentration and homogeneity).

Final results for all levels were within the acceptability limits stated in lab SOPs for concentration (80-120%) and homogeneity (CV < 10%). The formulations were found stable for 28 hours and 9 days at room temperature in the range from 25 to 125 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in lab SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity).

Duration of treatment / exposure:

Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, during pairing with females and thereafter through the day before necropsy performed on Day 37. Males were treated for a total of 36 days.
Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). One non pregnant female (Group 1) was dosed up to the day before necropsy.

Frequency of treatment:

Daily via oral gavage

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Medium

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Positive control:

Not a guideline requirement

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals. Observations of the cage tray were performed for all groups and were recorded up to daily intervals. This observation started from Day 0 post coitum until abnormalities were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture,
reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period, starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 4, 7 and 13 post partum starting from Day 1 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: For males and females blood samples were collected during the necropsy procedure, from the abdominal vena cava under isofluorane anaesthesia. The order of collection was equalised between groups.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes (although blood samples of a total of six females were inadvertantly collected without prior food deprivation. This was not believed to have impacted the results).
- How many animals: Collected by random selection from 5 males and 5 females from each group.
- Parameters checked: Haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leukocyte count (neutrophils, lymphocytes, easinophils, basophils, monocytes, large unstained cells, platelets). Prothrombin time and blood clotting time was also assessed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: For males and females blood samples were collected during the necropsy procedure, from the abdominal vena cava under isofluorane anaesthesia. The order of collection was equalised between groups.
- Animals fasted: Yes (although blood samples of a total of six females were inadvertantly collected without prior food deprivation. This was not believed to have impacted the results).
- How many animals: Collected by random selection from 5 males and 5 females from each group.
- Parameters checked: Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride and inorganic phosphorus.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: For males, the tests were performed on Day 34 or 36 of the study and for females on Day 12 post partum.
- Dose groups that were examined: 5 males and females from each group.
- Battery of functions tested: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order.

IMMUNOLOGY: Yes
- Time schedule for examinations: Blood samples taken as part of the scheduled necropsy procedure.
- How many animals: Samples from all males and pups on Day 14 post partum.
- Dose groups that were examined: All
- Parameters checked: T3, T4 and TSH samples were assayed by a multiplex assay using luminex magpix system and the milliplex map rat thyroid magnetic bead panel kit.

Sacrifice and pathology:

GROSS PATHOLOGY: All females were examined for external and internal abnormalities, number of visible implantation sites (pregnany animals) and number of corpora lutea (pregnant animals). All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed at Day 14 post partum were examined for external abnormalities and sex confirmation by gonads inspection.

HISTOPATHOLOGY: Yes. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. The PAS- H stained sections were used to identify the spermatogenic stages.
Parameters checked: abnormalities, adrenal glands, bone marrow (from sternum), brain (cerebrum, cerebellum, medula/pons), caecum, clitoral gland, colon, duodenum, epididymides, eyes, femur with joint, heart, ileum, jejunum (including peyer's patches), kidneys, liver, lungs (including bronchi), lymph nodes (cervical, mesenteric), mammary area, oesophagus, ovaries with oviducts, parathyroid glands, pituitary glands, penis, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus cervix and vagina.

The above tissues were examined in the first instance in 5 males and females randomly selected in the control and high dose group at terminal kill. All tissues were examined from animals killed or dying during the treatment period. All abnormalities were examined. Since differences were observed between control and high dose animals, the examinations were extended to the stomach in all remaining males and females and the thymus in all remaining females.

ORGAN WEIGHTS: From all animals completing the scheduled test period, organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
Parameters checked: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), duodenum, epididymides, heart, kidneys, liver, prostate gland (dorsolateral and ventral), seminal vesicles (with coagulating gland), spleen, testes, thymus, thyroid and uterus cervix.

Other examinations:

None specified

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: All males receiving 500 mg/kg/day (Group 4) showed salivation. This sign started to be observed before mating on Day 4 and thereafter during the mating phase. Hairloss on the head was also noted in a few males of the same group, during the study. One male receiving 250 mg/kg/day showed salivation for one day before mating (Day 7 of the study).

Females: Before the start of mating, salivation was noted in females receiving 500 mg/kg/day, as well as during the gestation phase. This sign was evident after a couple of days of treatment with similar incidence in both phases. In addition, one female receiving 500 mg/kg/day showed, on Day 1 of treatment, decreased activity, salivation and piloerection within 30 minutes of dosing. After 1.5 hour, decreased activity and piloerection were still present. After 24 hours from dosing,
all clinical signs disappeared. Convulsions were noted on the first day of dosing in one female receiving 100 mg/kg/day (Group 2). This sign appeared approximately after 30 minutes of dosing and disappeared shortly after. Approximately 1.5 hour after dosing, piloerection was recorded. After 24 hours from dosing, no signs were noted. Damaged eye noted in one control female and the mass on the mammary area noted in one female receiving 100 mg/kg/day were not considered treatment-related.

No clinical signs were noted during the post-partum phase.

During the pairing phase, when males and females were housed in the same cages, soft faeces were noted in control and treated groups. After the completion of the mating phase, soft faeces were noted in males of treated groups and sometimes also in the control group. During gestation and post partum phases, soft faeces were noted in females receiving the dose levels >/= 250 mg/kg/day and occasionally in those receiving 100 mg/kg/day. The above changes were in part due to the vehicle (a cathartic agent), rather than a direct effect of the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality occured throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences were seen in body weight of treated animals, when compared to controls throughout the study.
At the end of treatment (Day 22 of the mating phase), a statistically significant increase in body weight gain of treated males receiving the dose levels >/= 250 mg/kg/day was observed. Body weight gain of treated females remained quite comparable to the control group for the duration of the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was unaffected by treatment in both sexes during the study. On Day 4 post partum, a slight decrease in food consumption was noted in females receiving 100 mg/kg/day when compared to the control group. This change, even if significant at statistical analysis, was not considered treatment related since it was occasional and evident in only two females.

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):

Slight leucopenia was recorded in two males dosed at 500 mg/kg/day. Compared with mean control data, changes were 80% and 74%, respectively, and were mainly due to a decrease of lymphocytes. The other statistically significant differences between control and treated males (monocytes
and large unstained cells) were not dose-related, therefore they were considered to be unrelated to treatment.

No changes were recorded in coagulation parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Compared to controls, animals dosed at 500 mg/kg/day showed increases of bilirubin (2.5 fold in males and 8 fold in females), cholesterol (1.6 fold in males, 1.3 fold in females), triglycerides (1.4 fold in males, 2.4 in females).

It should be underlined that one female receiving 500 mg/kg/day showed a severe increase of triglycerides. In addition, males of the same treated group showed decreases of creatinine (20%), protein (6%) and globulin (12%), and females showed additional increase of glucose (1.5 fold). Animals receiving 250 mg/kg/day showed similar findings, such as: increase of bilirubin (females only, 5.5 fold), cholesterol (males only, 1.4 fold), triglycerides (1.3 fold in males and 1.5 fold in females), glucose (females only, 1.3 fold). The severity of the above findings observed was not considered to be suggestive of tissue/ organ injury and could reflect an organ adaptation. The statistically significant difference of alkaline phosphatase recorded between controls and males dosed at 250 mg/kg/day was not dose-related, therefore it was considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period were comparable between control and treated groups.

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males dosed at 500 mg/kg/day showed a slight increase of thyroxine (40% above controls). Due to the absence of other related changes, this finding was considered to be of no toxicological relevance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No changes were observed in terminal body weight of treated animals, when compared to the controls. A decrease in absolute (up to -29%) and relative (up to -25%) thymus weight was noted in all treated females when compared to the control group. This change was significant, at statistical analysis, only at 500 mg/kg/day for absolute weight. The above difference was accompanied by histopathological findings (atrophy) considered treatment-related. The remaining changes in absolute and/or relative organ weights, noted statistically significant in treated males and/or females sacrificed at term, could be comparable to control animals for histomorphology and therefore they are not considered toxicologically relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormal dark and/or red areas were observed in the stomach of high dose animals. This finding was more evident in females (6/10) than in males (1/10), when compared to the control group. Small thymus was observed in all treated female groups with a higher incidence in those receiving 500mg/kg/day, when compared to the control group. Only one high dose male showed the same finding. The macroscopic changes correlated with histopathological findings. All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related findings were seen in the stomach of high dose treated animals of both sexes and in the thymus of the high dose treated females:
Stomach: Focal or multifocal mucosal erosion of glandular region of the stomach was observed in few high dose males and females (3 out of 10) receiving 500 mg/kg/day, when compared to the controls. A similar change was also seen in one control female rat.

Thymus: Minimal to mild atrophy of thymus was observed in half of the high dose (5 out of 10) females receiving 500 mg/kg/day, when compared to the controls. This lesion was characterized by thymic lymphocyte depletion correlated with reduced thymus size and weight. The remaining sporadic lesions, including benign astrocytoma (neoplastic lesion confined mostly in the putamen and caudate nucleus of the brain) in one control male, or malignant adenocarcinoma of mammary gland in one low dose female, were considered to be an expression of spontaneous and/or incidental pathology seen in this species. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

other: Immune system and gastrointestinal tract

Organ:

stomach

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 250 mg/kg/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable OECD guideline study.

System:

other: Immune system and gastrointestinal tract

Organ:

stomach

thymus

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

Justification for classification or non-classification

Adverse effects to the thymus and stomach were noted on the available repeated dose/reproductive screen study (OECD 422). These have been considered in relation to classification under CLP for STOT RE endpoints.

The guidance values (effective dose causing an adverse effect) included in CLP to assist in classification are; Category 1 ≤ 30 mg/kg/day and Category 2 > 30 ≤ 300 mg/kg/day (for a sub-acute study).

 

In this particular situation the NOAEL falls below the cut off value (i.e. < 300 mg/kg/day) but the effective dose (essentially a LOAEL) is greater than this (i.e. 500 mg/kg/day). In this case ECHA guidance recommends using interpolation to make an assessment on whether a STOT RE classification is warranted and mentions that a worst case classification should be considered.

 

The effects in the thymus observed were limited to females (5/10) with no dose dependency (only observed in the high dose) and observed as mild/minimal. They also did not always correlate with macroscopic observations of a small thymus and vice versa and could have been a stress adaptation to the findings in the stomach. The effects in the stomach could also be considered as a result of the corrosivity of the substance.

However, from a conservative point of view classification as STOT RE Cat.2 is considered appropriate for this substance in relation to the thymus and stomach findings.